Plants having high plant map values

ABSTRACT

The present invention relates to novel plant expression constructs. More specifically the present invention provides DNA constructs comprising 5′ regulatory sequences for modulating the expression of operably linked genes in plants.

This application is a continuation of U.S. patent application Ser. No.10/427,169 filed May 1, 2003 (U.S. Pat. No. 6,919,495 issued July 19.2005), which is a divisional of U.S. patent application Ser. No.09/737,626 filed Dec. 15, 2000 (U.S. Pat. No. 6,660,911 issued Dec. 9,2003), which claimed priority to U.S. Provisional Patent ApplicationSer. No. 60/171,173 filed Dec. 16, 1999.

FIELD OF THE INVENTION

The present invention relates to the isolation and use of nucleic acidmolecules for control of gene expression in plants, specifically novelplant promoters.

BACKGROUND OF THE INVENTION

One of the goals of plant genetic engineering is to produce plants withagronomically important characteristics or traits. Recent advances ingenetic engineering have provided the requisite tools to producetransgenic plants that contain and express foreign genes (Kahl et al.,World J. of Microbiol. Biotech. 11:449–460, 1995). Particularlydesirable traits or qualities of interest for plant genetic engineeringwould include but are not limited to resistance to insects, fungaldiseases, and other pests and disease-causing agents, tolerances toherbicides, enhanced stability or shelf-life, yield, environmentaltolerances, and nutritional enhancements. The technological advances inplant transformation and regeneration have enabled researchers to takeexogenous DNA, such as a gene or genes from a heterologous or a nativesource, and incorporate the exogenous DNA into the plant's genome. Inone approach, expression of a novel gene that is not normally expressedin a particular plant or plant tissue may confer a desired phenotypiceffect. In another approach, transcription of a gene or part of a genein an antisense orientation may produce a desirable effect by preventingor inhibiting expression of an endogenous gene.

In order to produce a transgenic plant, a construct that includes aheterologous gene sequence that confers a desired phenotype whenexpressed in the plant is introduced into a plant cell. The constructalso includes a plant promoter that is operably linked to theheterologous gene sequence, often a promoter not normally associatedwith the heterologous gene. The construct is then introduced into aplant cell to produce a transformed plant cell, and the transformedplant cell is regenerated into a transgenic plant. The promoter controlsexpression of the introduced DNA sequence to which the promoter isoperably linked and thus affects the desired characteristic conferred bythe DNA sequence.

It would be advantageous to have a variety of promoters to tailor geneexpression such that a gene or gene(s) is transcribed efficiently at theright time during plant growth and development, in the optimal locationin the plant, and in the amount necessary to produce the desired effect.For example, constitutive expression of a gene product may be beneficialin one location of the plant but less beneficial in another part of theplant. In other cases, it may be beneficial to have a gene productproduced at a certain developmental stage of the plant or in response tocertain environmental or chemical stimuli. The commercial development ofgenetically improved germplasm has also advanced to the stage ofintroducing multiple traits into crop plants, often referred to as agene stacking approach. In this approach, multiple genes conferringdifferent characteristics of interest can be introduced into a plant. Itis important when introducing multiple genes into a plant that each geneis modulated or controlled for optimal expression and that theregulatory elements are diverse in order to reduce the potential of genesilencing. In light of these and other considerations, it is apparentthat optimal control of gene expression and regulatory element diversityare important in plant biotechnology.

SUMMARY OF THE INVENTION

The present invention relates to DNA plant expression constructs thatcomprise Arabidopsis actin (Act) promoter sequences Act1a, Act1b, Act 2,Act3, Act7, Act8, Act11, Act12 and the elongation factor 1α (EF1α)promoter sequence, and fragments and cis elements derived from thesepromoters operably linked to heterologous structural gene sequences thatfunction in crop plant cells.

Thus, according to one embodiment of the invention, a recombinant DNAconstruct is provided that comprises, in operable linkage, a promoterthat is functional in a cell of a crop plant, the promoter comprising:at least one cis element derived from SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26; a structural DNA sequence heterologous to thepromoter; and a 3′ non-translated region that functions in plants tocause the addition of polyadenylated nucleotides to the 3′ end of theRNA sequence. For example, the promoter may consist essentially of a 5′regulatory region derived from any of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26 (including or excluding any intron sequenceslocated therein). The structural gene may comprise any heterologousnucleotide sequence wherein expression of the sequence results in anagronomically useful trait or product in a transgenic crop plant.

According to another aspect of the invention is a DNA constructcomprising a structural DNA sequence operably linked to the promotersequences of the present invention that encode a protein employed toconfer herbicide tolerance to a crop plant. This herbicide toleranceprotein includes, but is not limited to glyphosate tolerance proteingenes such as a glyphosate resistant EPSP synthase gene alone, or incombination with one or more glyphosate degrading protein genes.

According to another embodiment of the invention, DNA constructs such asthose described above are provided wherein the promoter is a hybrid orchimeric promoter comprising at least one cis element derived from oneor more of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26operably linked to a heterologous promoter sequence such as acaulimovirus promoter, for example the Cauliflower mosaic virus 35Spromoter or the Figwort mosaic virus promoter.

According to another embodiment of the invention, DNA constructs, suchas those described above, are provided in tandem, wherein the promoteris a hybrid or chimeric promoter comprising at least one cis elementderived from one or more of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, andSEQ ID NO:26 operably linked to a heterologous gene sequence thatexpresses in transgenic crop plant cells. The chimeric promotersequences more specifically comprising the sequences identified in SEQID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

According to another embodiment of the invention, a DNA construct suchas that described above is provided wherein the structural DNA sequenceis a glyphosate tolerance gene, such that when the DNA construct isintroduced into a plant cell, it confers to the plant cell tolerance toan aqueous glyphosate formulation that includes at least 50 grams acidequivalent per liter (g a.e./l) of glyphosate. In other relatedembodiments, the DNA construct confers to the plant cell tolerance toglyphosate formulations having higher glyphosate concentrations (forexample, at least 300 grams acid equivalent per liter of glyphosate.According to one embodiment, the DNA construct confers to the plant celltolerance to at least one application of Roundup Ultra® at a rate of 16ounces (oz) per acre, for example, and in other embodiments, glyphosatetolerance extends to one to two or more applications of 16 oz per acre,32 oz per acre, or 64 oz per acre, for example.

According to another embodiment of the invention, transgenic crop plantsare provided that are transformed with a DNA construct as describedabove, including monocot species and dicot species. We have discoveredthat the Arabidopsis actin and Arabidopsis EF1α promoters aresufficiently active in other crop plant species such as cotton, tomato,and sunflower, for example, that when used to control expression of aglyphosate tolerance gene, such as aroA:CP4, the plants toleratecommercial application rates of glyphosate, exhibiting good vegetativetolerance and low damage to reproductive tissues. Such promoters canalso be used to express other genes of interest in plants, including,but not limited to, genes that confer herbicide tolerance, insectcontrol, disease resistance, increased stability or shelf, higher yield,nutritional enhancement, expression of a pharmaceutical or other desiredpolypeptide product, or a desirable change in plant physiology ormorphology, and so on.

According to another embodiment of the invention, transgenic crop plantsare provided that are transformed with multiple DNA constructscomprising the Arabidopsis actin and Arabidopsis EF1α promoters aresufficiently active in other plant species such as cotton, tomato,sunflower, for example, that when used to control expression of aglyphosate tolerance gene such as aroA:CP4, the plants toleratedcommercial application rates of glyphosate, exhibiting good vegetativetolerance and low damage to reproductive tissues. Such promoters canalso be used to express other genes of interest in plants, including,but not limited to, genes that confer herbicide tolerance, insectcontrol, disease resistance, increased stability or shelf, higher yield,nutritional enhancement, expression of a pharmaceutical or other desiredpolypeptide product, or a desirable change in plant physiology ormorphology, and so on.

According to another embodiment of the invention, transgenic crop plantsare provided that are transformed with DNA constructs comprising theArabidopsis actin and Arabidopsis EF1α promoters as chimeric DNAmolecules in fusion with caulimovirus DNA molecules having promoteractivity in plants sufficiently active in other plant species such ascotton, tomato, canola, soybean, and sunflower, for example, that whenused to control expression of a glyphosate tolerance gene such asaroA:CP4, the plants tolerate commercial application rates ofglyphosate, exhibiting good vegetative tolerance and low damage toreproductive tissues. Such promoters can also be used to express othergenes of interest in plants, including, but not limited to, genes thatconfer herbicide tolerance, insect control, disease resistance,increased stability or shelf, higher yield, nutritional enhancement,expression of a pharmaceutical or other desired polypeptide product, ora desirable change in plant physiology or morphology, and so on.

According to another embodiment of the invention methods are providedfor expressing a structural DNA sequence in a plant. Such methodscomprise, providing a DNA construct as described above, introducing theDNA construct into a plant cell, and regenerating the plant cell toproduce a plant such that the structural DNA is expressible in theplant. According to a related embodiment, a method of controlling weedsis provided in which the DNA construct comprises a glyphosate tolerancegene and one applies to a crop plant transformed with the DNA constructan amount of glyphosate that is sufficient to control weeds withoutsignificantly damaging the crop plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map of pCGN8086

FIG. 2 is a plasmid map of pMON45325

FIG. 3 is a plasmid map of pMON45331

FIG. 4 is a plasmid map of pMON45332

FIG. 5 is a plasmid map of pCGN9190

FIG. 6 is a plasmid map of pCGN9153

FIG. 7 is a plasmid map of pCGN8099

FIG. 8 is a plasmid map of pCGN8088

FIG. 9 is a plasmid map of pCGN8068

FIG. 10 is a plasmid map of pCGN8096

FIG. 11 is a plasmid map of pCGN9151

FIG. 12 is a plasmid map of pMON10156

FIG. 13 is a plasmid map of pMON52059

FIG. 14 is a plasmid map of pMON54952

FIG. 15 is a plasmid map of pMON54953

FIG. 16 is a plasmid map of pMON54954

FIG. 17 is a plasmid map of pMON54955

FIG. 18 is a plasmid map of pMON54956

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

-   SEQ ID NO:1 is the forward PCR primer used for the isolation of the    Act2 promoter-   SEQ ID NO:2 is the reverse PCR primer used for the isolation of the    Act2 promoter-   SEQ ID NO:3 is the forward PCR primer used for the isolation of the    Act8 promoter-   SEQ ID NO:4 is the reverse PCR primer used for the isolation of the    Act8 promoter-   SEQ ID NO:5 is the forward PCR primer used for the isolation of the    Act11 promoter-   SEQ ID NO:6 is the reverse PCR primer used for the isolation of the    Act11 promoter-   SEQ ID NO:7 is the forward PCR primer used for the isolation of the    EF1 promoter-   SEQ ID NO:8 is the reverse PCR primer used for the isolation of the    EF1 promoter-   SEQ ID NO:9 is the sequence of the Act2 promoter including the    intron sequence of the Act2 gene. Base positions 1–764 represent the    promoter sequence; base positions 765–1215 represent the intron    followed by 5 bases of 5′ untranslated region (5′ UTR) prior to the    ATG; the transcription start site is located at base position 597.-   SEQ ID NO:10 is the sequence of the Act8 promoter including the    first intron of the Act8 gene. Base positions 1–797 represent the    promoter sequence; base positions 798–1259 represent the intron    followed by 10 bases of 5′ UTR prior to the ATG; the transcription    start site is located at base position 646.-   SEQ ID NO:11 is the sequence of the ActI 1 promoter including the    first intron of the Act11 gene. Base positions 1–1218 represent the    promoter sequence; base positions 1219–1381 represent the intron    followed by 10 bases of 5′ UTR prior to the ATG; the transcription    start site is located at base position 1062.-   SEQ ID NO:12 is the sequence of the EF1 promoter including the first    intron of the EF1 gene. Base positions 1–536 represent the promoter    sequence; base positions 537–1137 represent the intron followed by    22 bases of 5′ UTR prior to the ATG; the transcription start site is    located at base position 481.-   SEQ ID NO:13 is the forward PCR primer used for the isolation of the    Act1a promoter-   SEQ ID NO:14 is the forward PCR primer used for the isolation of the    Act1b promoter-   SEQ ID NO:15 is the reverse PCR primer used for the isolation of the    Act1a and Act1b promoter-   SEQ ID NO:16 is the forward PCR primer used for the isolation of the    Act3 promoter-   SEQ ID NO:17 is the reverse PCR primer used for the isolation of the    Act3 promoter-   SEQ ID NO:18 is the forward PCR primer used for the isolation of the    Act7 promoter-   SEQ ID NO:19 is the reverse PCR primer used for the isolation of the    Act7 promoter-   SEQ ID NO:20 is the forward PCR primer used for the isolation of the    Act12 promoter-   SEQ ID NO:21 is the reverse PCR primer used for the isolation of the    Act12 promoter-   SEQ ID NO:22 is the sequence of the Act1a promoter including the    first intron of the Act1a gene. Base positions 1–1033 represent the    promoter sequence; base positions 1034–1578 represent the intron and    5′ UTR.-   SEQ ID NO:23 is the sequence of the Act1b promoter including the    first intron of the Act1b gene. Base positions 1–914 represent the    promoter sequence; base positions 915–1468 represent the intron and    5′ UTR sequence.-   SEQ ID NO:24 is the sequence of the Act3 promoter including the    first intron of the Act3 gene. Base positions 1–1023 represent the    promoter sequence; base positions 1024–1642 represent the intron and    5′ UTR sequence.-   SEQ ID NO:25 is the sequence of the Act7 promoter including the    first intron of the Act7 gene. Base positions 1–600 represent the    promoter sequence; base positions 601–1241 represent the intron and    5′ UTR sequence.-   SEQ ID NO:26 is the sequence of the Act12 promoter including the    first intron of the Act12 gene. Base positions 1–1017 represent the    promoter sequence; base positions 1018–1313 represent the intron and    5′ UTR sequence.-   SEQ ID NO:27 is the sequence of the chimeric FMV-Act11 promoter    including the first intron of the Act11 gene. Base positions 1–536    represent the FMV promoter sequence; base positions 553–1946    represent the Arabidopsis Actin 11 promoter, intron and 5′ UTR    sequence.-   SEQ ID NO:28 is the sequence of the chimeric FMV-EF1α promoter    including the first intron of the EF1α gene. Base positions 1–536    represent the FMV promoter sequence; base positions 553–1695    represent the EF1α promoter, intron, and 5′ UTR sequence.-   SEQ ID NO:29 is the sequence of the CaMV-Act8 promoter including the    first intron of the Act8 gene. Base positions 1–523 present the CaMV    promoter sequence; base positions 534–1800 represent the Act8    promoter, intron and 5′ UTR sequence.-   SEQ ID NO:30 is the sequence of the CaMV-Act2 promoter including the    first intron of the Act2 gene. Base positions 1–523 represent the    CaMV promoter sequence; base positions 534–1742 represent the Act2    promoter, intron and 5′ UTR sequence.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define, andto guide those of ordinary skill in the art in the practice of, thepresent invention. Unless otherwise noted, terms are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. The nomenclature for DNA bases as set forth at 37 CFR §1.822 is used. The standard one- and three-letter nomenclature for aminoacid residues is used.

“Nucleic acid (sequence)” or “polynucleotide (sequence)” refers tosingle- or double-stranded DNA or RNA of genomic or synthetic origin,i.e., a polymer of deoxyribonucleotide or ribonucleotide bases,respectively, read from the 5′ (upstream) end to the 3′ (downstream)end. The nucleic acid can represent the sense or complementary(antisense) strand.

“Native” refers to a naturally occurring (“wild-type”) nucleic acidsequence.

“Heterologous” sequence refers to a sequence which originates from aforeign source or species or, if from the same source, is modified fromits original form.

An “isolated” nucleic acid sequence is substantially separated orpurified away from other nucleic acid sequences with which the nucleicacid is normally associated in the cell of the organism in which thenucleic acid naturally occurs, i.e., other chromosomal orextrachromosomal DNA. The term embraces nucleic acids that arebiochemically purified so as to substantially remove contaminatingnucleic acids and other cellular components. The term also embracesrecombinant nucleic acids and chemically synthesized nucleic acids. Theterm “substantially purified”, as used herein, refers to a moleculeseparated from other molecules normally associated with it in its nativestate. More preferably, a substantially purified molecule is thepredominant species present in a preparation. A substantially purifiedmolecule may be greater than 60% free, preferably 75% free, morepreferably 90% free from the other molecules (exclusive of solvent)present in the natural mixture. The term “substantially purified” is notintended to encompass molecules present in their native state.

A first nucleic acid sequence displays “substantially identity” to areference nucleic acid sequence if, when optimally aligned (withappropriate nucleotide insertions or deletions totaling less than 20percent of the reference sequence over the window of comparison) withthe other nucleic acid (or its complementary strand), there is at leastabout 75% nucleotide sequence identity, preferably at least about 80%identity, more preferably at least about 85% identity, and mostpreferably at least about 90% identity over a comparison window of atleast 20 nucleotide positions, preferably at least 50 nucleotidepositions, more preferably at least 100 nucleotide positions, and mostpreferably over the entire length of the first nucleic acid. Optimalalignment of sequences for aligning a comparison window may be conductedby the local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482, 1981; by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988;preferably by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA) in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis. Thereference nucleic acid may be a full-length molecule or a portion of alonger molecule. Alternatively, two nucleic acids are have substantialidentity if one hybridizes to the other under stringent conditions, asdefined below.

A first nucleic acid sequence is “operably linked” with a second nucleicacid sequence when the sequences are so arranged that the first nucleicacid sequence affects the function of the second nucleic-acid sequence.Preferably, the two sequences are part of a single contiguous nucleicacid molecule and more preferably are adjacent. For example, a promoteris operably linked to a gene if the promoter regulates or mediatestranscription of the gene in a cell.

A “recombinant” nucleic acid is made by an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques. Techniques for nucleic-acid manipulation arewell-known (see for example Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, 1989; Mailga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995;Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997), volume2, Detecting Genes, (1998), volume 3, Cloning Systems, (1999) volume 4,Mapping Genomes, (1999), Cold Spring Harbor, N.Y.).

Methods for chemical synthesis of nucleic acids are discussed, forexample, in Beaucage and Carruthers, Tetra. Letts. 22:1859–1862, 1981,and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemicalsynthesis of nucleic acids can be performed, for example, on commercialautomated oligonucleotide synthesizers.

A “synthetic nucleic acid sequence” can be designed and chemicallysynthesized for enhanced expression in particular host cells and for thepurposes of cloning into appropriate constructs. Host cells oftendisplay a preferred pattern of codon usage (Murray et al., 1989).Synthetic DNAs designed to enhance expression in a particular hostshould therefore reflect the pattern of codon usage in the host cell.Computer programs are available for these purposes including but notlimited to the “BestFit” or “Gap” programs of the Sequence AnalysisSoftware Package, Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis. 53711.

“Amplification” of nucleic acids or “nucleic acid reproduction” refersto the production of additional copies of a nucleic acid sequence and iscarried out using polymerase chain reaction (PCR) technologies. Avariety of amplification methods are known in the art and are described,inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCRProtocols: A Guide to Methods and Applications, ed. Innis et al.,Academic Press, San Diego, 1990. In PCR, a primer refers to a shortoligonucleotide of defined sequence which is annealed to a DNA templateto initiate the polymerase chain reaction.

“Transformed”, “transfected”, or “transgenic” refers to a cell, tissue,organ, or organism into which has been introduced a foreign nucleicacid, such as a recombinant construct. Preferably, the introducednucleic acid is integrated into the genomic DNA of the recipient cell,tissue, organ or organism such that the introduced nucleic acid isinherited by subsequent progeny. A “transgenic” or “transformed” cell ororganism also includes progeny of the cell or organism and progenyproduced from a breeding program employing such a “transgenic” plant asa parent in a cross and exhibiting an altered phenotype resulting fromthe presence of a recombinant construct or construct.

The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA, syntheticDNA, or other DNA that encodes a peptide, polypeptide, protein, or RNAmolecule, and regions flanking the coding sequence involved in theregulation of expression. Some genes can be transcribed into mRNA andtranslated into polypeptides (structural genes); other genes can betranscribed into RNA (e.g. rRNA, tRNA); and other types of gene functionas regulators of expression (regulator genes).

“Expression” of a gene refers to the transcription of a gene to producethe corresponding mRNA and translation of this mRNA to produce thecorresponding gene product, i.e., a peptide, polypeptide, or protein.Gene expression is controlled or modulated by regulatory elementsincluding 5′ regulatory elements such as promoters.

“Genetic component” refers to any nucleic acid sequence or geneticelement which may also be a component or part of an expressionconstruct. Examples of genetic components include, but are not limitedto promoter regions, 5′ untranslated leaders, introns, genes, 3′untranslated regions, and other regulatory sequences or sequences whichaffect transcription or translation of one or more nucleic acidsequences.

The terms “recombinant DNA construct”, “recombinant construct”,“expression construct” or “expression cassette” refer to any agent suchas a plasmid, cosmid, virus, BAC (bacterial artificial chromosome),autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence,derived from any source, capable of genomic integration or autonomousreplication, comprising a DNA molecule in which one or more DNAsequences have been linked in a functionally operative manner usingwell-known recombinant DNA techniques.

“Complementary” refers to the natural association of nucleic acidsequences by base-pairing (A-G-T pairs with the complementary sequenceT-C-A). Complementarity between two single-stranded molecules may bepartial, if only some of the nucleic acids pair are complementary; orcomplete, if all bases pair are complementary. The degree ofcomplementarity affects the efficiency and strength of hybridization andamplification reactions.

“Homology” refers to the level of similarity between nucleic acid oramino acid sequences in terms of percent nucleotide or amino acidpositional identity, respectively, i.e., sequence similarity oridentity. Homology also refers to the concept of similar functionalproperties among different nucleic acids or proteins.

“Promoter” refers to a nucleic acid sequence located upstream or 5′ to atranslational start codon of an open reading frame (or protein-codingregion) of a gene and that is involved in recognition and binding of RNApolymerase II and other proteins (trans-acting transcription factors) toinitiate transcription. A “plant promoter” is a native or non-nativepromoter that is functional in plant cells. Constitutive promoters arefunctional in most or all tissues of a plant throughout plantdevelopment. Tissue-, organ- or cell-specific promoters are expressedonly or predominantly in a particular tissue, organ, or cell type,respectively. Rather than being expressed “specifically” in a giventissue, organ, or cell type, a promoter may display “enhanced”expression, i.e., a higher level of expression, in one part (e.g., celltype, tissue, or organ) of the plant compared to other parts of theplant. Temporally regulated promoters are functional only orpredominantly during certain periods of plant development or at certaintimes of day, as in the case of genes associated with circadian rhythm,for example. Inducible promoters selectively express an operably linkedDNA sequence in response to the presence of an endogenous or exogenousstimulus, for example by chemical compounds (chemical inducers) or inresponse to environmental, hormonal, chemical, and/or developmentalsignals. Inducible or regulated promoters include, for example,promoters regulated by light, heat, stress, flooding or drought,phytohormones, wounding, or chemicals such as ethanol, jasmonate,salicylic acid, or safeners.

Any plant promoter can be used as a 5′ regulatory sequence formodulating expression of a particular gene or genes. One preferredpromoter would be a plant RNA polymerase II promoter. Plant RNApolymerase II promoters, like those of other higher eukaryotes, havecomplex structures and are comprised of several distinct elements. Onesuch element is the TATA box or Goldberg-Hogness box, which is requiredfor correct expression of eukaryotic genes in vitro and accurate,efficient initiation of transcription in vivo. The TATA box is typicallypositioned at approximately −25 to −35, that is, at 25 to 35 basepairs(bp) upstream (5′) of the transcription initiation site, or cap site,which is defined as position +1 (Breathnach and Chambon, Ann. Rev.Biochem. 50:349–383, 1981; Messing et al., In: Genetic Engineering ofPlants, Kosuge et al., eds., pp. 211–227, 1983). Another common element,the CCAAT box, is located between −70 and −100 bp. In plants, the CCAATbox may have a different consensus sequence than the functionallyanalogous sequence of mammalian promoters (the plant analogue has beentermed the “AGGA box” to differentiate it from its animal counterpart;Messing et al., In: Genetic Engineering of Plants, Kosuge et al., eds.,pp. 211–227, 1983). In addition, virtually all promoters includeadditional upstream activating sequences or enhancers (Benoist andChambon, Nature 290:304–310, 1981; Gruss et al., Proc. Nat. Acad. Sci.USA 78:943–947, 1981; and Khoury and Gruss, Cell 27:313–314, 1983)extending from around −100 bp to −1,000 bp or more upstream of thetranscription initiation site.

When fused to heterologous DNA sequences, such promoters typically causethe fused sequence to be transcribed in a manner that is similar to thatof the gene sequence with which the promoter is normally associated.Promoter fragments that include regulatory sequences can be added (forexample, fused to the 5′ end of, or inserted within, an active promoterhaving its own partial or complete regulatory sequences (Fluhr et al.,Science 232:1106–1112, 1986; Ellis et al., EMBO J. 6:11–16, 1987;Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986–8990, 1987;Poulsen and Chua, Mol. Gen. Genet. 214:16–23, 1988; Comai et al., PlantMol. Biol. 15:373–381, 1991). Alternatively, heterologous regulatorysequences can be added to the 5′ upstream region of an inactive,truncated promoter, e.g., a promoter including only the core TATA and,sometimes, the CCAAT elements (Fluhr et al., Science 232:1106–1112,1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986–8990,1987; Aryan et al., Mol. Gen. Genet. 225:65–71, 1991).

Promoters are typically comprised of multiple distinct “cis-actingtranscriptional regulatory elements,” or simply “cis-elements,” each ofwhich confers a different aspect of the overall control of geneexpression (Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986–8990, 1987; Ellis et al., EMBO J. 6:11–16, 1987; Benfey et al.,EMBO J. 9:1677–1684, 1990). “Cis elements” bind trans-acting proteinfactors that regulate transcription. Some cis elements bind more thanone factor, and trans-acting transcription factors may interact withdifferent affinities with more than one cis element (Johnson andMcKnight, Ann. Rev. Biochem. 58:799–839, 1989). Plant transcriptionfactors, corresponding cis elements, and analysis of their interactionare discussed, for example, In: Martin, Curr. Opinions Biotech.7:130–138, 1996; Murai, In: Methods in Plant Biochemistry and MolecularBiology, Dashek, ed., CRC Press, 1997, pp. 397–422; and Methods in PlantMolecular Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995,pp. 233–300. The promoter sequences of the present invention can contain“cis elements” that confer or modulate gene expression.

Cis elements can be identified by a number of techniques, includingdeletion analysis, i.e., deleting one or more nucleotides from the 5′end or internal to a promoter; DNA binding protein analysis using DnaseI footprinting, methylation interference, electrophoresis mobility-shiftassays, in vivo genomic footprinting by ligation-mediated PCR and otherconventional assays; or by sequence similarity with known cis elementmotifs by conventional sequence comparison methods. The fine structureof a cis element can be further studies by mutagenesis (or substitution)of one or more nucleotides of the element or by other conventionalmethods (see for example, Methods in Plant Biochemistry and MolecularBiology, Dashek, ed., CRC Press, 1997, pp. 397–422; and Methods in PlantMolecular Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995,pp. 233–300).

Cis elements can be obtained by chemical synthesis or by cloning frompromoters that include such elements. Cis elements can also besynthesized with additional flanking sequences that contain usefulrestriction enzyme sites to facilitate subsequence manipulation. In oneembodiment, the promoters are comprised of multiple distinct“cis-elements”. In a preferred embodiment sequence regions comprising“cis elements” of the nucleic acid sequences of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26 are identified using computerprograms including, but not limited to MEME or SIGNALSCAN that aredesigned specifically to identify cis elements, or domains or motifswithin sequences.

The present invention includes fragments or cis elements of SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 or homologues of ciselements known to effect gene regulation that show homology with thenucleic acid sequences of the present invention. Such nucleic acidfragments can include any region of the disclosed sequences. Thepromoter regions or partial promoter regions of the present invention asshown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 cancontain at least one regulatory element including, but not limited to“cis elements” or domains that are capable of regulating expression ofoperably linked DNA sequences, such as in male reproductive tissues.

Plant promoters can also include promoters produced through themanipulation of known promoters to produce synthetic, chimeric, orhybrid promoters. Such promoters can also combine cis elements from oneor more promoters, for example, by adding a heterologous regulatorysequence to an active promoter with its own partial or completeregulatory sequences (Ellis et al., EMBO J. 6:11–16, 1987; Strittmatterand Chua, Proc. Nat. Acad. Sci. USA 84:8986–8990, 1987; Poulsen andChua, Mol. Gen. Genet. 214:16–23, 1988; Comai et al., Plant. Mol. Biol.15:373–381, 1991). Chimeric promoters have also been developed by addinga heterologous regulatory sequence to the 5′ upstream region of aninactive, truncated promoter, i.e., a promoter that includes only thecore TATA and, optionally, the CCAAT elements (Fluhr et al., Science232:1106–1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986–8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65–71, 1991).

Chimeric or hybrid promoters according to the present invention mayinclude at least one known cis element such as elements that areregulated by numerous environmental factors such as light, heat, orstress; elements that are regulated or induced by pathogens orchemicals, and the like. Such elements may either positively ornegatively regulate gene expression, depending on the conditions.Examples of cis elements include, but are not limited to oxygenresponsive elements (Cowen et al., J. Biol. Chem. 268(36):26904, 1993),light regulatory elements (see for example, Bruce and Quail, Plant Cell2: 1081, 1990, and Bruce et al., EMBO J. 10:3015, 1991, a cis elementreponsive to methyl jasmonate treatment (Beaudoin and Rothstein, PlantMol. Biol. 33:835, 1997, salicylic acid-responsive elements (Strange etal., Plant J. 11: 1315, 1997, heat shock response elements (Pelham etal., Trends Genet. 1:31, 1985, elements responsive to wounding andabiotic stress (Loace et al., Proc. Natl. Acad. Sci. U.S.A. 89:9230,1992; Mhiri et al., Plant Mol. Biol. 33:257, 1997), cold-responsiveelements (Baker et al., Plant Mol. Biol. 24:701, 1994; Jiang et al.,Plant Mol. Biol. 30:679, 1996; Nordin et al., Plant Mol. Biol. 21:641,1993; Zhou et al., J. Biol. Chem. 267:23515, 1992), and droughtresponsive elements, (Yamaguchi et al., Plant Cell 6:251–264, 1994; Wanget al., Plant Mol. Biol. 28:605, 1995; Bray E. A. Trends in PlantScience 2:48, 1997).

In another embodiment, the nucleotide sequences as shown in SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQID NO:29, and SEQ ID NO:30 includes any length of said nucleotidesequences that is capable of regulating an operably linked DNA sequence.For example, the sequences as disclosed in SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, andSEQ ID NO:30 may be truncated or have portions deleted and still becapable of regulating transcription of an operably linked DNA sequence.In a related embodiment, a cis element of the disclosed sequences mayconfer a particular specificity such as conferring enhanced expressionof operably linked DNA sequences in certain tissues. Consequently, anysequence fragments, portions, or regions of the disclosed sequences ofSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQID NO:28, SEQ ID NO:29, and SEQ ID NO:30 can be used as regulatorysequences including but not limited to cis elements or motifs of thedisclosed sequences. For example, one or more base pairs may be deletedfrom the 5′ or 3′ end of a promoter sequence to produce a “truncated”promoter. One or more base pairs can also be inserted, deleted, orsubstituted internally to a promoter sequence. Promoters can beconstructed such that promoter fragments or elements are operablylinked, for example, by placing such a fragment upstream of a minimalpromoter. A minimal or basal promoter is a piece of DNA which is capableof recruiting and binding the basal transcription machinery. One exampleof basal transcription machinery in eukaryotic cells is the RNApolymerase II complex and its accessory proteins. The enzymaticcomponents of the basal transcription machinery are capable ofinitiating and elongating transcription of a given gene, utilizing aminimal or basal promoter. That is, there are not added cis-actingsequences in the promoter region that are capable of recruiting andbinding transcription factors that modulate transcription, e.g.,enhance, repress, render transcription hormone-dependent, etc.Substitutions, deletions, insertions or any combination thereof can becombined to produce a final construct.

The promoter sequences of the present invention may be modified, forexample for expression in other plant systems. In another approach,novel hybrid promoters can be designed or engineered by a number ofmethods. Many promoters contain upstream sequences which activate,enhance or define the strength and/or specificity of the promoter(Atchison, Ann. Rev. Cell Biol. 4:127, 1988). T-DNA genes, for examplecontain “TATA” boxes defining the site of transcription initiation andother upstream elements located upstream of the transcription initiationsite modulate transcription levels (Gelvin, In: Transgenic Plants (Kung,S.-D. and Us, R., eds, San Diego: Academic Press, pp. 49–87, 1988).Another chimeric promoter combined a trimer of the octopine synthase(ocs) activator to the mannopine synthase (mas) activator plus promoterand reported an increase in expression of a reporter gene (Min Ni etal., The Plant Journal 7:661, 1995). The upstream regulatory sequencesof the present invention can be used for the construction of suchchimeric or hybrid promoters. Methods for construction of variantpromoters of the present invention include but are not limited tocombining control elements of different promoters or duplicatingportions or regions of a promoter (see for example U.S. Pat. Nos.5,110,732 and 5,097,025). Those of skill in the art are familiar withthe specific conditions and procedures for the construction,manipulation and isolation of macromolecules (e.g., DNA molecules,plasmids, etc.), generation of recombinant organisms and the screeningand isolation of genes, (see for example Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, 1989; Mailga etal., Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995;Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997), volume2, Detecting Genes, (1998), volume 3, Cloning Systems, (1999) volume 4,Mapping Genomes, (1999), Cold Spring Harbor, N.Y.).

The design, construction, and use of chimeric or hybrid promoterscomprising one or more of cis elements of SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26, for modulating or regulating the expression ofoperably linked nucleic acid sequences are also encompassed by thepresent invention.

The promoter sequences, fragments, regions or cis elements thereof ofSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQID NO:28, SEQ ID NO:29, and SEQ ID NO:30 are capable of transcribingoperably linked DNA sequences in multiple tissues and therefore canselectively regulate expression of genes in multiple tissues.

For a number of agronomic traits, transcription of a gene or genes ofinterest is desirable in multiple tissues to confer the desiredcharacteristic(s). The availability of suitable promoters that regulatetranscription of operably linked genes in selected target tissues ofinterest is desirable, since it may not be desirable to express a genein every tissue, but only in certain tissues. For example, if onedesires to selectively express a target gene for expression of gene forherbicide tolerance, one may desire expression of the herbicidetolerance gene in vegetative and reproductive tissues. The promotersequences of the present invention are useful for regulating geneexpression in multiple tissues including, but not limited to rapidlygrowing meristematic tissues, male reproductive tissues (androecium)such as pollen, anthers, and filaments, and female reproductive tissues(gynoecium) such as the stigma, style, and ovaries, leaves, sepals, andpetals. The promoters of the present invention therefore have utilityfor expression of herbicide tolerance genes, for example, wheretolerance is desired in multiple tissues and stages of plantdevelopment. The promoter sequences of the present invention haveutility for regulating transcription of any target gene including butnot limited to genes for control of fertility, yield, insect tolerance,fungal tolerance, herbicide tolerance, or any desirable trait ofinterest. Particularly preferred genes include herbicide tolerance genesor insect tolerance genes.

In one embodiment, the promoters of the present invention haveparticular utility for regulating expression of an herbicide tolerancegene where expression of a gene is desired in multiple tissues. Forexample, the herbicide tolerance gene may confer tolerance to theherbicide glyphosate. Examples of suitable glyphosate tolerance genesinclude, but are not limited to glyphosate resistant5-enolpyruvyl-3-phosphoshikimate synthase (hereinafter referred to asEPSP synthase or EPSPS) genes or gene products that degrade glyphosatesuch as, a glyphosate oxidoreductase and phosphonate N-acetyltransferase. It is important to have a wide variety of choices of 5′regulatory elements for any plant biotechnology strategy in order tohave suitable regulatory elements that are most efficient for theexpression profile desired.

In another embodiment, the promoters of the present invention haveutility for determining gene function. The function of many genes isunknown and the promoters of the present invention can be used asgenetic elements in a construct to allow a phenotypic evaluation of oneor more genes expressed in a sense or antisense orientation. Thepromoters of the present invention can be components in a plantexpression construct developed for a high throughput assay where highlevels of gene expression in constitutive and reproductive tissues isdesired.

Any plant can be selected for the identification of genes and regulatorysequences. Examples of suitable plant targets for the isolation of genesand regulatory sequences would include but are not limited to alfalfa,apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado,banana, barley, beans, beet, blackberry, blueberry, broccoli, brusselssprouts, cabbage, canola, cantaloupe, carrot, cassaya, castorbean,cauliflower, celery, cherry, chicory, cilantro, citrus, clementines,clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir,eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd,grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon,lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut,oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamentalplant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper,persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato,pumpkin, quince, radiata pine, radiscchio, radish, rapeseed, raspberry,rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry,sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea,tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat,yams, and zucchini. Particularly preferred plants for the identificationof regulatory sequences are Arabidopsis, corn, wheat, soybean, andcotton.

The promoter sequences of the present invention were isolated fromArabidopsis thaliana plant DNA. In a preferred embodiment, a constructincludes the promoter sequences of the present invention operably linkedto a transcribable sequence along with suitable terminator andregulatory elements. Such a construct may be transformed into a suitabletarget plant of interest. Any plant can be used as a suitable host fornucleic acid constructs comprising the promoter sequences of the presentinvention. Examples of suitable target plants of interest would include,but are not limited to alfalfa, broccoli, cabbage, canola, cauliflower,corn, cotton, cranberry, cucumber, lettuce, pea, poplar, pine, potato,onion, rice, raspberry, soybean, sugarcane, sugarbeet, sunflower,tomato, and wheat.

Promoter Isolation and Modification Methods

Any number of methods can be used to isolate fragments of the promotersequences disclosed herein. A PCR-based approach can be used to amplifyflanking regions from a genomic library of a plant using publiclyavailable sequence information. A number of methods are known to thoseof skill in the art to amplify unknown DNA sequences adjacent to a coreregion of known sequence. Methods include but are not limited to inversePCR (IPCR), vectorette PCR, Y-shaped PCR and genome walking approaches.For the present invention, the nucleic acid molecules were isolated fromArabidopsis by designing PCR primers based on available sequenceinformation.

Nucleic acid fragments can also be obtained by other techniques such asby directly synthesizing the fragment by chemical means, as is commonlypracticed by using an automated oligonucleotide synthesizer. Fragmentscan also be obtained by application of nucleic acid reproductiontechnology, such as the PCR (polymerase chain reaction) technology byrecombinant DNA techniques generally known to those of skill in the artof molecular biology. Regarding the amplification of a targetnucleic-acid sequence (e.g., by PCR) using a particular amplificationprimer pair, “stringent PCR conditions” refer to conditions that permitthe primer pair to hybridize only to the target nucleic-acid sequence towhich a primer having the corresponding wild-type sequence (or itscomplement) would bind and preferably to produce a unique amplificationproduct.

Those of skill in the art are aware of methods for the preparation ofplant genomic DNA. In one approach, genomic DNA libraries can beprepared from a chosen species by partial digestion with a restrictionenzyme and size selecting the DNA fragments within a particular sizerange. The genomic DNA can be cloned into a suitable construct includingbut not limited to a bacteriophage, and prepared using a suitableconstruct such as a bacteriophage using a suitable cloning kit from anynumber of vendors (see for example Stratagene, La Jolla Calif. or GibcoBRL, Gaithersburg, Md.).

In another embodiment, the nucleotide sequences of the promotersdisclosed herein can be modified. Those skilled in the art can createDNA molecules that have variations in the nucleotide sequence. Thenucleotide sequences of the present invention as shown in SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28,SEQ ID NO:29, and SEQ ID NO:30 may be modified or altered to enhancetheir control characteristics. For example, the sequences may bemodified by insertion, deletion or replacement of template sequences ina PCR-based DNA modification approach. “Variant” DNA molecules are DNAmolecules containing changes in which one or more nucleotides of anative sequence is deleted, added, and/or substituted, preferably whilesubstantially maintaining promoter function. In the case of a promoterfragment, “variant” DNA can include changes affecting the transcriptionof a minimal promoter to which it is operably linked. Variant DNAmolecules can be produced, for example, by standard DNA mutagenesistechniques or by chemically synthesizing the variant DNA molecule or aportion thereof.

In addition to their use in modulating gene expression, the promotersequences of the present invention also have utility as probes orprimers in nucleic acid hybridization experiments. The nucleic-acidprobes and primers of the present invention can hybridize understringent conditions to a target DNA sequence. The term “stringenthybridization conditions” is defined as conditions under which a probeor primer hybridizes specifically with a target sequence(s) and not withnon-target sequences, as can be determined empirically. The term“stringent conditions” is functionally defined with regard to thehybridization of a nucleic-acid probe to a target nucleic acid (i.e., toa particular nucleic-acid sequence of interest) by the specifichybridization procedure (see for example Sambrook et al., 1989, at9.52–9.55, Sambrook et al., 1989 at 9.47–9.52, 9.56–9.58; Kanehisa,Nucl. Acids Res. 12:203–213, 1984; and Wetmur and Davidson, J. Mol.Biol. 31:349–370, 1968). Appropriate stringency conditions which promoteDNA hybridization are, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in laboratory manualsincluding but not limited to Current Protocols in Molecular Biology,John Wiley & Sons, N.Y., 1989, 6.3.1–6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or either the temperature or the salt concentration may be heldconstant while the other variable is changed. For example, hybridizationusing DNA or RNA probes or primers can be performed at 65° C. in 6×SSC,0.5% SDS, 5× Denhardt's, 100 μg/mL nonspecific DNA (e.g., sonicatedsalmon sperm DNA) with washing at 0.5×SSC, 0.5% SDS at 65° C., for highstringency.

A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if they exhibit complete complementarity. As usedherein, molecules are said to exhibit “complete complementarity” whenevery nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low stringency” conditions. Similarly, the moleculesare said to be “complementary” is they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high stringency” conditions. It is contemplated thatlower stringency hybridization conditions such as lower hybridizationand/or washing temperatures can be used to identify related sequenceshaving a lower degree of sequence similarity if specificity of bindingof the probe or primer to target sequence(s) is preserved. Accordingly,the nucleotide sequences of the present invention can be used for theirability to selectively form duplex molecules with complementarystretches of DNA fragments. Detection of DNA segments via hybridizationis well-known to those of skill in the art, and thus depending on theapplication envisioned, one will desire to employ varying hybridizationconditions to achieve varying degrees of selectivity of probe towardstarget sequence and the method of choice will depend on the desiredresults. Conventional stringency conditions are described in Sambrook,et al., Molecular Cloning, A Laboratory Manual, 2^(nd) Ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989, and by Haymes et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C., 1985.

In one embodiment of the present invention, the nucleic acid sequencesSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, or a fragment,region, cis element, or oligomer of these sequences, are used inhybridization assays of other plant tissues to identify closely relatedor homologous genes and associated regulatory sequences. These includebut are not limited to Southern or northern hybridization assays on anysubstrate including but not limited to an appropriately prepared planttissue, cellulose, nylon, or combination filter, chip, or glass slide.Such methodologies are well known in the art and are available in a kitor preparation which can be supplied by commercial vendors.

A fragment of a nucleic acid as used herein is a portion of the nucleicacid that is less than full-length. For example, for the presentinvention any length of nucleotide sequence that is less than thedisclosed nucleotide sequences of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26 is considered to be a fragment. A fragment canalso comprise at least a minimum length capable of hybridizingspecifically with a native nucleic acid under stringent hybridizationconditions as defined above. The length of such a minimal fragment ispreferably at least 8 nucleotides, more preferably 15 nucleotides, evenmore preferably at least 20 nucleotides, and most preferably at least 30nucleotides of a native nucleic acid sequence.

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. “Primers” areisolated nucleic acids that are annealed to a complementary target DNAstrand by nucleic acid hybridization to form a hybrid between the primerand the target DNA strand, then extended along the target DNA strand bya polymerase, e.g., a DNA polymerase. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other conventional nucleic-acid amplification methods.

Probes and primers are generally 11 nucleotides or more in length,preferably 18 nucleotides or more, more preferably 25 nucleotides, andmost preferably 30 nucleotides or more. Such probes and primershybridize specifically to a target DNA or RNA sequence under highstringency hybridization conditions and hybridize specifically to atarget native sequence of another species under lower stringencyconditions. Preferably, probes and primers according to the presentinvention have complete sequence similarity with the native sequence,although probes differing from the native sequence and that retain theability to hybridize to target native sequences may be designed byconventional methods. Methods for preparing and using probes and primersare described, for example, in Molecular Cloning: A Laboratory Manual,2nd ed., vol. 1–3, ed. Sambrook et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al.,1989”); Current Protocols in Molecular Biology, ed. Ausubel et al.,Greene Publishing and Wiley-Interscience, New York, 1992 (with periodicupdates) (hereinafter, “Ausubel et al., 1992); and Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press: SanDiego, 1990. PCR-primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, © 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.). Primers and probes based on the nativepromoter sequences disclosed herein can be used to confirm and, ifnecessary, to modify the disclosed sequences by conventional methods,e.g., by re-cloning and re-sequencing.

Constructs and Expression Constructs

Native or synthetic nucleic acids according to the present invention canbe incorporated into recombinant nucleic acid constructs, typically DNAconstructs, capable of introduction into and replication in a host cell.In a preferred embodiment, the nucleotide sequences of the presentinvention as shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 orfragments, variants or derivatives thereof are incorporated into anexpression cassette which includes the promoter regions of the presentinvention operably linked to a genetic component such as a selectable,screenable, or scorable marker gene.

In another embodiment, the disclosed nucleic acid sequences of thepresent invention as shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30 are operably linked to a genetic component such as a nucleic acidwhich confers a desirable characteristic associated with plantmorphology, physiology, growth and development, yield, nutritionalenhancement, disease or pest resistance, or environmental or chemicaltolerance. These genetic components such as marker genes or agronomicgenes of interest can function in the identification of a transformedplant cell or plant, or a produce a product of agronomic utility.

In another embodiment, one genetic component produces a product whichserves as a selection device and functions in a regenerable plant tissueto produce a compound which would confer upon the plant tissueresistance to an otherwise toxic compound. Genes of interest for use asa selectable, screenable, or scorable marker would include but are notlimited to GUS (coding sequence for beta-glucuronidase), GFP (codingsequence for green fluorescent protein), LUX (coding gene forluciferase), antibiotic resistance marker genes, or herbicide tolerancegenes. Examples of transposons and associated antibiotic resistancegenes include the transposons Tns (bla), Tn5 (nptII), Tn7 (dhfr),penicillins, kanamycin (and neomycin, G418, bleomycin); methotrexate(and trimethoprim); chloramphenicol; kanamycin and tetracycline.

Characteristics useful for selectable markers in plants have beenoutlined in a report on the use of microorganisms (Advisory Committee onNovel Foods and Processes, July 1994). These include stringent selectionwith minimum number of nontransformed tissues, large numbers ofindependent transformation events with no significant interference withthe regeneration, application to a large number of species, andavailability of an assay to score the tissues for presence of themarker.

A number of selectable marker genes are known in the art and severalantibiotic resistance markers satisfy these criteria, including thoseresistant to kanamycin (nptII), hygromycin B (aph IV) and gentamycin(aac3 and aacC4). Useful dominant selectable marker genes include genesencoding antibiotic resistance genes (e.g., resistance to hygromycin,kanamycin, bleomycin, G418, streptomycin or spectinomycin); andherbicide resistance genes (e.g., phosphinothricin acetyltransferase). Auseful strategy for selection of transformants for herbicide resistanceis described, e.g., in Vasil, Cell Culture and Somatic Cell Genetics ofPlants, Vols. I–III, Laboratory Procedures and Their ApplicationsAcademic Press, New York, 1984. Particularly preferred selectable markergenes for use in the present invention would genes which conferresistance to compounds such as antibiotics like kanamycin, andherbicides like glyphosate (Della-Cioppa et al., Bio/Technology 5(6),1987, U.S. Pat. Nos. 5,463,175, 5,633,435). Other selection devices canalso be implemented and would still fall within the scope of the presentinvention.

For the practice of the present invention, conventional compositions andmethods for preparing and using DNA constructs and host cells areemployed, as discussed, inter alia, in Sambrook et al., 1989. In apreferred embodiment, the host cell is a plant cell. A number of DNAconstructs suitable for stable transfection of plant cells or for theestablishment of transgenic plants have been described in, e.g., Pouwelset al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987);Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990; and R. R. D. Croy Plant Molecular BiologyLabFax, BIOS Scientific Publishers, 1993. Plant expression constructscan include, for example, one or more cloned plant genes under thetranscriptional control of 5′ and 3′ regulatory sequences. They can alsoinclude a selectable marker as described to select for host cellscontaining the expression construct. Such plant expression constructsalso contain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and apolyadenylation signal. Other sequences of bacterial origin are alsoincluded to allow the construct to be cloned in a bacterial host. Theconstruct will also typically contain a broad host range prokaryoticorigin of replication. In a particularly preferred embodiment, the hostcell is a plant cell and the plant expression construct comprises apromoter region as disclosed in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30; an operably linked transcribable sequence; and a transcriptiontermination sequence. Other regulatory sequences envisioned as geneticcomponents in an expression construct include but is not limited tonon-translated leader sequence which can be coupled with the promoter.In a particularly preferred embodiment, the host cell is a plant celland the plant expression construct comprises a promoter region asdisclosed in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNOS:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; an operably linkedtranscribable sequence, and a transcription termination sequence. Plantexpression constructs also can comprise additional sequences includingbut not limited to polylinker sequences that contain restriction enzymesites that are useful for cloning purposes.

Genetic Elements in Plant Expression Constructs

Plant expression constructs may include more than one expressible genesequence, each operably linked to a different promoter. A number ofpromoters have utility for plant gene expression for any gene ofinterest including but not limited to selectable markers, scorablemarkers, genes for pest tolerance, disease tolerance, nutritionalenhancements and any other gene of agronomic interest. Examples ofconstitutive promoters useful for plant gene expression include but arenot limited to, the cauliflower mosaic virus (CAMV) P-35S promoter,which confers constitutive, high-level expression in most plant tissues(see, e.g., Odel et al., Nature 313:810, 1985), including monocots (see,e.g., Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto,Mol. Gen. Genet. 220:389, 1990); a tandemly duplicated version of theCaMV 35S promoter, the enhanced 35S promoter (P-e35S) the nopalinesynthase promoter (An et al., Plant Physiol. 88:547, 1988), the octopinesynthase promoter (Fromm et al., Plant Cell 1:977, 1989); and thefigwort mosaic virus (P-FMV) promoter as described in U.S. Pat. No.5,378,619 and an enhanced version of the FMV promoter (P-eFMV) where thepromoter sequence of P-FMV is duplicated in tandem, the cauliflowermosaic virus 19S promoter, a sugarcane bacilliform virus promoter, acommelina yellow mottle virus promoter, and other plant DNA viruspromoters known to express in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can beused for expression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat (Callis et al., Plant Physiol. 88:965,1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., PlantCell 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpson etal., EMBO J. 4:2723, 1985), (3) hormones, such as abscisic acid(Marcotte et al., Plant Cell 1:969, 1989), (4) wounding (e.g., wunl,Siebertz et al., Plant Cell 1:961, 1989); or (5) chemicals such asmethyl jasmonate, salicylic acid, or Safener. It may also beadvantageous to employ (6) organ-specific promoters (e.g., Roshal etal., EMBO J. 6:1155, 1987; Schernthaner et al., EMBO J. 7:1249, 1988;Bustos et al., Plant Cell 1:839, 1989). The promoters of the presentinvention are plant promoters that are capable of transcribing operablylinked DNA sequences in rapidly growing meristematic tissue andreproductive tissues and can be operably linked to any gene of interestin an expression construct.

Plant expression constructs can include RNA processing signals, e.g.,introns, which may be positioned upstream or downstream of apolypeptide-encoding sequence in the transgene. In addition, theexpression constructs may include additional regulatory sequences fromthe 3′-untranslated region of plant genes (Thornburg et al., Proc. Natl.Acad. Sci. USA 84:744 (1987); An et al., Plant Cell 1:115 (1989), e.g.,a 3′ terminator region to increase mRNA stability of the mRNA, such asthe PI-II terminator region of potato or the octopine or nopalinesynthase 3′ terminator regions. 5′ non-translated regions of a mRNA canplay an important role in translation initiation and can also be agenetic component in a plant expression construct. For example,non-translated 5′ leader sequences derived from heat shock protein geneshave been demonstrated to enhance gene expression in plants (see, forexample U.S. Pat. No. 5,362,865). These additional upstream anddownstream regulatory sequences may be derived from a source that isnative or heterologous with respect to the other elements present on theexpression construct.

The promoter sequences of the present invention are used to control geneexpression in plant cells. The disclosed promoter sequences are geneticcomponents that are part of constructs used in plant transformation. Thepromoter sequences of the present invention can be used with anysuitable plant transformation plasmid or construct containing aselectable or screenable marker and associated regulatory elements, asdescribed, along with one or more nucleic acids expressed in a mannersufficient to confer a particular desirable trait. Examples of suitablestructural genes of agronomic interest envisioned by the presentinvention would include but are not limited to one or more genes forinsect tolerance, such as a Bacillus thuringiensis (B.t.) gene, pesttolerance such as genes for fungal disease control, herbicide tolerancesuch as genes conferring glyphosate tolerance, and genes for qualityimprovements such as yield, nutritional enhancements, environmental orstress tolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s). For example, structuralgenes would include any gene that confers insect tolerance including butnot limited to a Bacillus insect control protein gene as described in WO9931248, herein incorporated by reference in its entirety, U.S. Pat. No.5,689,052, herein incorporated by reference in its entirety, U.S. Pat.Nos. 5,500,365 and 5,880,275, herein incorporated by reference it theirentirety. In another embodiment, the structural gene can confertolerance to the herbicide glyphosate as conferred by genes including,but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPSgene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, hereinincorporated by reference in its entirety, or glyphosate oxidoreductasegene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporatedby reference in its entirety.

Alternatively, the DNA coding sequences can effect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev. 9:207,1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillitoe, Mol.Biotech. 7:125,1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

In addition to regulatory elements or sequences located upstream (5′) orwithin a DNA sequence, there are downstream (3′) sequences that affectgene expression. Thus, the term regulatory sequence as used hereinrefers to any nucleotide sequence located upstream, within, ordownstream to a DNA sequence which controls, mediates, or affectsexpression of a gene product in conjunction with the protein syntheticapparatus of the cell.

Those of skill in the art are aware of the constructs suitable for planttransformation. The promoter sequences of the present invention arepreferably incorporated into an expression construct using screenable orscorable markers as described and tested in transient analyses toprovide an indication of gene expression in transformed plants. Methodsof testing gene expression in transient assays are known to those ofskill in the art. Transient expression of marker genes has been reportedusing a variety of plants, tissues and DNA delivery systems. Forexample, types of transient analyses can include but are not limited todirect gene delivery via electroporation or particle bombardment oftissues in any transient plant assay using any plant species ofinterest. Such transient systems would include but are not limited toprotoplasts from suspension cultures in wheat (Zhou et al., Plant CellReports 12:612. 1993), electroporation of leaf protoplasts of wheat(Sethi et al., J. Crop Sci. 52: 152, 1983); electroporation ofprotoplast prepared from corn tissue (Sheen, J. The Plant Cell 3: 225,1991), or particle bombardment of specific tissues of interest. Thepresent invention encompasses the use of any transient expression systemto evaluate regulatory sequences operatively linked to selected reportergenes, marker genes or agronomic genes of interest. Examples of planttissues envisioned to test in transients via an appropriate deliverysystem would include but are not limited to leaf base tissues, callus,cotyledons, roots, endosperm, embryos, floral tissue, pollen, andepidermal tissue.

Any scorable or screenable marker can be used in a transient assay.Preferred marker genes for transient analyses of the promoters or 5′regulatory sequences of the present invention include a β-glucuronidase(GUS) gene or a green fluorescent protein (GFP) gene. The expressionconstructs containing the 5′ regulatory sequences operably linked to amarker gene are delivered to the tissues and the tissues are analyzed bythe appropriate mechanism, depending on the marker. The quantitative orqualitative analyses are used as a tool to evaluate the potentialexpression profile of the 5′ regulatory sequences when operativelylinked to genes of agronomic interest in stable plants. Ultimately, the5′ regulatory sequences of the present invention are directlyincorporated into suitable plant transformation expression constructscomprising the 5′ regulatory sequences operatively linked to atranscribable DNA sequence interest, transformed into plants and thestably transformed plants and progeny thereof analyzed for the desiredexpression profile conferred by the 5′ regulatory sequences.

Suitable expression constructs for introducing exogenous DNA into plantcells would include but are not limited to disarmed Ti-plasmids forAgrobacterium-mediated methods. These constructs can contain aresistance marker, 1-2 T-DNA borders, and origins of replication for E.coli and Agrobacterium along with one or more genes of interest andassociated regulatory regions. Those of skill in the art are aware thatfor Agrobacterium-mediated approaches a number of strains and methodsare available. Such strains would include but are not limited toAgrobacterium strains C58, LBA4404, EHA101 and EHA105. Particularlypreferred strains are Agrobacterium tumefaciens strains.

Exemplary nucleic acids which may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences whichoriginate with or are present in the same species, but are incorporatedinto recipient cells by genetic engineering methods rather thanclassical reproduction or breeding techniques. However, the term“exogenous” is also intended to refer to genes that are not normallypresent in the cell being transformed, or perhaps simply not present inthe form, structure, etc., as found in the transforming DNA segment orgene, or genes which are normally present and that one desires toexpress in a manner that differs from the natural expression pattern,e.g., to over-express. Thus, the term “exogenous” gene or DNA isintended to refer to any gene or DNA segment that is introduced into arecipient cell, regardless of whether a similar gene may already bepresent in such a cell. The type of DNA included in the exogenous DNAcan include DNA which is already present in the plant cell, DNA fromanother plant, DNA from a different organism, or a DNA generatedexternally, such as a DNA sequence containing an antisense message of agene, or a DNA sequence encoding a synthetic or modified version of agene.

The plant transformation constructs containing the promoter sequences ofthe present invention may be introduced into plants by any planttransformation method. Several methods are available for introducing DNAsequences into plant cells and are well known in the art. Suitablemethods include but are not limited to bacterial infection (e.g., withAgrobacterium as described above), binary bacterial artificialchromosome constructs, direct delivery of DNA (e.g. via PEG-mediatedtransformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers), andacceleration of DNA coated particles (reviewed in Potrykus, Ann. Rev.Plant Physiol. Plant Mol. Biol., 42: 205, 1991).

Methods for specifically transforming dicots primarily use Agrobacteriumtumefaciens. For example, transgenic plants reported include but are notlimited to cotton (U.S. Pat. Nos 5,004,863; 5,159,135; 5,518,908, WO97/43430), soybean (U.S. Pat. Nos. 5,569,834; 5,416,011; McCabe et al.,Bio/Technology, 6:923, 1988; Christou et al., Plant Physiol., 87:671,1988); Brassica (U.S. Pat. No. 5,463,174), and peanut (Cheng et al.,Plant Cell Rep., 15: 653, 1996).

Similar methods have been reported in the transformation of monocots.Transformation and plant regeneration using these methods have beendescribed for a number of crops including but not limited to asparagus(Asparagus officinalis; Bytebier et al., Proc. Natl. Acad. Sci. U.S.A.,84: 5345, 1987); barley (Hordeum vulgarae; Wan and Lemaux, PlantPhysiol., 104: 37, 1994); maize (Zea mays; Rhodes, C. A., et al.,Science, 240: 204, 1988; Gordon-Kamm, et al., Plant Cell, 2: 603, 1990;Fromm, et al., Bio/Technology, 8: 833, 1990; Koziel, et al.,Bio/Technology, 11: 194, 1993); oats (Avena sativa; Somers, et al.,Bio/Technology, 10: 1589, 1992); orchardgrass (Dactylis glomerata; Horn,et al., Plant Cell Rep., 7: 469, 1988); rice (Oryza sativa, includingindica and japonica varieties, Toriyama, et al., Bio/Technology, 6: 10,1988; Zhang, et al., Plant Cell Rep., 7: 379, 1988; Luo and Wu, PlantMol. Biol. Rep., 6: 165, 1988; Zhang and Wu, Theor. Appl. Genet., 76:835, 1988; Christou, et al., Bio/Technology, 9: 957, 1991); sorghum(Sorghum bicolor; Casas, A. M., et al., Proc. Natl. Acad. Sci. U.S.A.,90: 11212, 1993); sugar cane (Saccharum spp.; Bower and Birch, Plant J.,2: 409, 1992); tall fescue (Festuca arundinacea; Wang, Z. Y. et al.,Bio/Technology, 10: 691, 1992); turfgrass (Agrostis palustris; Zhong etal., Plant Cell Rep., 13: 1, 1993); wheat (Triticum aestivum; Vasil etal., Bio/Technology, 10: 667, 1992; Weeks T., et al., Plant Physiol.,102: 1077, 1993; Becker, et al., Plant, J. 5: 299, 1994), and alfalfa(Masoud, S. A., et al., Transgen. Res., 5: 313, 1996). It is apparent tothose of skill in the art that a number of transformation methodologiescan be used and modified for production of stable transgenic plants fromany number of target crops of interest.

Plant Analysis Methods

The transformed plants are analyzed for the presence of the genes ofinterest and the expression level and/or profile conferred by thepromoter sequences of the present invention. Those of skill in the artare aware of the numerous methods available for the analysis oftransformed plants. A variety of methods are used to assess geneexpression and determine if the introduced gene(s) is integrated,functioning properly, and inherited as expected. For the presentinvention the promoters can be evaluated by determining the expressionlevels of genes to which the promoters are operatively linked. Apreliminary assessment of promoter function can be determined by atransient assay method using reporter genes, but a more definitivepromoter assessment can be determined from the analysis of stableplants. Methods for plant analysis include but are not limited toSouthern blots or northern blots, PCR-based approaches, biochemicalanalyses, phenotypic screening methods, field evaluations, andimmunodiagnostic assays.

The methods of the present invention including but not limited to PCRtechnologies, genomic DNA isolation, expression construct construction,transient assays, and plant transformation methods are well known tothose of skill in the art and are carried out using standard techniquesor modifications thereof.

Glyphosate Spray Tests

In one embodiment a greenhouse or field evaluation for glyphosatetolerance is conducted. The term “glyphosate” is used herein to refercollectively to the parent herbicide N-phosphonomethylglycine (otherwiseknown as glyphosate acid), to a salt or ester thereof, or to a compoundwhich is converted to N-phosphonomethylglycine in plant tissues or whichotherwise provides N-phosphonomethylglycine in ionic form (otherwiseknown as glyphosate ion). Illustratively, water-soluble glyphosate saltsuseful herein are disclosed in U.S. Pat. Nos. 3,799,758 and 4,405,531 toFranz, the disclosure of which is incorporated herein by reference.Glyphosate salts that can be used according to the present inventioninclude but are not restricted to alkali metal, for example sodium andpotassium, salts; ammonium salt; C₁₋₁₆ alkylammonium, for exampledimethylammonium and isopropylammonium, salts; C₁₋₁₆ alkanolammonium,for example monoethanolammonium, salt; C₁₋₁₆ alkylsulfonium, for exampletrimethylsulfonium, salts; mixtures thereof and the like. The glyphosateacid molecule has three acid sites having different pKa values;accordingly mono-, di- and tribasic salts, or any mixture thereof, orsalts of any intermediate level of neutralization, can be used.

Glyphosate salts are commercially significant in part because they arewater-soluble. Many ammonium, alkylammonium, alkanolammonium,alkylsulfonium and alkali metal salts are highly water-soluble, allowingfor formulation as highly concentrated aqueous solutions which can bediluted in water at the point of use.

Such concentrated aqueous solutions can contain about 50 to about 500grams per liter of glyphosate, expressed as acid equivalent (g a.e./l).Higher glyphosate concentrations, for example about 300 to about 500 ga.e./l, are preferred.

Glyphosate salts are alternatively formulated as water-soluble orwater-dispersible compositions, in the form for example of powders,granules, pellets or tablets. Such compositions are often known as dryformulations, although the term “dry” should not be understood in thiscontext to imply the complete absence of water. Typically, dryformulations contain less than about 5% by weight of water, for exampleabout 0.5% to about 2% by weight of water. Such formulations areintended for dissolution or dispersion in water at the point of use.

Contemplated dry glyphosate formulations can contain about 5% to about80% by weight of glyphosate, expressed as acid equivalent (% a.e.).Higher glyphosate concentrations within the above range, for exampleabout 50% to about 80% a.e., are preferred. Especially useful salts ofglyphosate for making dry formulations are sodium and ammonium salts.

Plant treatment compositions and liquid and dry concentrate compositionsof the invention can optionally contain one or more desired excipientingredients. Especially useful excipient ingredients for glyphosatecompositions are surfactants, which assist in retention of aqueous spraysolutions on the relatively hydrophobic surfaces of plant leaves, aswell as helping the glyphosate to penetrate the waxy outer layer(cuticle) of the leaf and thereby contact living tissues within theleaf. Surfactants can perform other useful functions as well.

There is no restriction in the type or chemical class of surfactant thatcan be used in glyphosate compositions of the invention. Nonionic,anionic, cationic and amphoteric types, or combinations of more than oneof these types, are all useful in particular situations. However, it isgenerally preferred that at least one of the surfactants, if any,present should be other than anionic; i.e., at least one of thesurfactants should be nonionic, cationic or amphoteric.

Many surfactants useful herein have a chemical structure that comprisesone or more moieties each consisting of a single C₂₋₄ alkylene oxideunit or a polymerized or copolymerized chain of C₂₋₄ alkylene oxideunits. Such surfactants are referred to as polyoxyalkylene surfactantsand include nonionic, anionic, cationic and amphoteric types.Polyoxyalkylene surfactants useful in presently contemplatedcompositions contain about 2 to about 100 C₂₋₄ alkylene oxide units. Inpreferred polyoxyalkylene surfactants the alkylene oxide units form oneor more chains of either ethylene oxide or copolymerized ethylene oxideand propylene oxide, each chain of alkylene oxide units having aterminal hydrido group or a C₁₋₄ alkyl or C₁₋₄ alkanoyl end-cap.

Hydrophobic moieties of surfactants useful in compositions of theinvention can be essentially hydrocarbon based, in which case thehydrophobic moieties are typically C₈₋₂₄, preferably C₁₂₋₁₈, alkyl,alkenyl, alkylaryl, alkanoyl or alkenoyl chains. These chains can belinear or branched. Alternatively, the hydrophobic moieties can containsilicon atoms, for example in the form of siloxane groups such asheptamethyltrisiloxane groups, or fluorine atoms, for example aspartially-fluorinated alkyl or perfluoroalkyl chains.

Among nonionic surfactants, especially preferred classes includepolyoxyethylene alkyl, alkenyl or alkylaryl ethers, such as ethoxylatedprimary or secondary alcohols or alkylphenols, polyoxyethylene alkyl oralkenyl esters, such as ethoxylated fatty acids, polyoxyethylenesorbitan alkyl esters, glyceryl alkyl esters, sucrose esters, alkylpolyglycosides, and the like. Representative specific examples of suchnonionic surfactants include polyoxyethylene (9) nonylphenol, Neodol™25-7 of Shell (a polyoxyethylene (7) C₁₂₋₁₅ linear primary alcohol),Tergitol™ 15-S-9 of Union Carbide (a polyoxyethylene (9) C₁₂₋₁₅secondary alcohol), Tween™ 20 of ICI (a polyoxyethylene (20) sorbitanmonolaurate) and Agrimul™ PG-2069 of Henkel (a C₉₋₁₁ alkylpolyglucoside).

Among cationic surfactants, especially preferred classes includepolyoxyethylene tertiary alkylamines or alkenylamines, such asethoxylated fatty amines, quaternary ammonium surfactants,polyoxyethylene alkyletheramines, and the like. Representative specificexamples of such cationic surfactants include polyoxyethylene (5)cocoamine, polyoxyethylene (15) tallowamine, distearyldimethylammoniumchloride, cetyltrimethylammonium bromide, methylbis(2-hydroxyethyl)cocoammonium chloride, N-dodecylpyridine chloride andpolyoxypropylene (8) ethoxytrimethylammonium chloride. Particularlypreferred polyoxyethylene alkyletheramines are those disclosed in PCTPublication No. WO 96/32839. Many cationic quaternary ammoniumsurfactants of diverse structures are known in the art to be useful incombination with glyphosate and can be used in compositions contemplatedherein; such quaternary ammonium surfactants have the formula(NR^(a)R^(b)R^(c)R^(d))_(m)A_(n)where A is a suitable anion such as chloride, bromide, iodide, acetate,sulfate or phosphate, m and n are integers such that the positiveelectrical charges on cations (NR^(a)R^(b)R^(c)R^(d)) balance thenegative electrical charges on anions A, and options for R^(a), R^(b),R^(c) and R^(d) include, without limitation:

-   -   (i) R^(a) is benzyl or C₈₋₂₄, preferably C₁₂₋₁₈, alkyl or        alkenyl, and R^(b), R^(c) and R^(d) are independently C₁₋₄        alkyl, preferably methyl;    -   (ii) R^(a) and R^(b) are independently C₈₋₂₄, preferably C₁₂₋₁₈,        alkyl or alkenyl, and R^(c) and R^(d) are independently C₁₋₄        alkyl, preferably methyl;    -   (iii) R^(a) is C₈₋₂₄, preferably C₁₂₋₁₈, alkyl or alkenyl, R^(b)        is a polyoxyalkylene chain having about 2 to about 100 C₂₋₄        alkylene oxide units, preferably ethylene oxide units, and R^(c)        and R^(d) are independently C₁₋₄ alkyl, preferably methyl;    -   (iv) R^(a) is C₈₋₂₄, preferably C₁₂₋₁₈, alkyl or alkenyl, R^(b)        and R^(c) are polyoxyalkylene chains having in total about 2 to        about 100 C₂₋₄ alkylene oxide units, preferably ethylene oxide        units, and R^(d) is C₁₋₄ alkyl, preferably methyl; or    -   (v) R^(a) is a polyoxyalkylene chain having about 2 to about 100        C₂₋₄ alkylene oxide units in which C₃₋₄ alkylene oxide units,        preferably propylene oxide units, predominate and R^(b), R^(c)        and R^(d) are independently C₁₋₄ alkyl, preferably methyl or        ethyl. Particularly preferred quaternary ammonium surfactants of        this type are those disclosed in U.S. Pat. No. 5,464,807 to        Claude et al.

In one embodiment, the anion A associated with such a quaternaryammonium surfactant can be a glyphosate anion.

Among amphoteric surfactants, including as is customary in the artsurfactants more correctly described as zwitterionic, especiallypreferred classes include polyoxyethylene alkylamine oxides,alkylbetaines, alkyl-substituted amino acids and the like.Representative examples of such amphoteric surfactants includedodecyldimethylamine oxide, polyoxyethylene (2) cocoamine oxide andstearyldimethylbetaine.

Standard reference sources from which one of skill in the art can selectsuitable surfactants, without limitation to the above mentioned classes,include Handbook of Industrial Surfactants, Second Edition (1997)published by Gower, McCutcheon 's Emulsifiers and Detergents, NorthAmerican and International Editions (1997) published by MC PublishingCompany, and International Cosmetic Ingredient Dictionary, Sixth Edition(1995) Volumes 1 and 2, published by the Cosmetic, Toiletry andFragrance Association.

Other optional components of compositions of the invention includeagents to modify color, viscosity, gelling properties, freezing point,hygroscopicity, caking behavior, dissolution rate, dispersibility, orother formulation characteristics.

Examples of commercial formulations of glyphosate include, withoutrestriction, those sold by Monsanto Company as ROUNDUP®, ROUNDUP® ULTRA,ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE,RODEO®, POLARIS®, SPARK® and ACCORD®) herbicides, all of which containglyphosate as its isopropylammonium salt; those sold by Monsanto Companyas ROUNDUP® DRY and RIVAL® herbicides, which contain glyphosate as itsammonium salt; that sold by Monsanto Company as ROUNDUP® GEOFORCE, whichcontains glyphosate as its sodium salt; and that sold by Zeneca Limitedas TOUCHDOWN® (herbicide, which contains glyphosate as itstrimethylsulfonium salt.

The selection of application rates for a glyphosate formulation that arebiologically effective is within the skill of the ordinary agriculturaltechnician. One of skill in the art will likewise recognize thatindividual plant conditions, weather conditions and growing conditionscan affect the results achieved in practicing the process of the presentinvention. Over two decades of glyphosate use and published studiesrelating to such use have provided abundant information from which aweed control practitioner can select glyphosate application rates thatare herbicidally effective on particular species at particular growthstages in particular environmental conditions.

A process of the present invention is applicable to any and all plantspecies on which glyphosate is biologically effective as a herbicide orplant growth regulator. This encompasses a very wide variety of plantspecies worldwide. Likewise, compositions of the invention can beapplied to any and all plant species on which glyphosate is biologicallyeffective.

In one embodiment, a glyphosate-containing herbicide is applied to theplant comprising the DNA constructs of the present invention, and theplants are evaluated for tolerance to the glyphosate herbicide. Anyformulation of glyphosate can be used for testing plants comprising theDNA constructs of the present invention. For example, a glyphosatecomposition such as Roundup Ultra™ can be used. The testing parametersfor an evaluation of the glyphosate tolerance of the plant will varydepending on a number of factors. Factors would include, but are notlimited to the type of glyphosate formulation, the concentration andamount of glyphosate used in the formulation, the type of plant, theplant developmental stage during the time of the application,environmental conditions, the application method, and the number oftimes a particular formulation is applied. For example, plants can betested in a greenhouse environment using a spray application method. Thetesting range using Roundup Ultra™ can include, but is not limited to 8oz/acre to 256 oz/acre. The preferred commercially effective range canbe from 16 oz/acre to 64 oz/acre of Roundup Ultra™, depending on thecrop and stage of plant development. A crop can be sprayed with at leastone application of a glyphosate formulation. For testing in cotton anapplication of 32 oz/acre at the 3-leaf stage may be followed byadditional applications at later stages in development. For wheat anapplication of 32 oz/acre of Roundup Ultra™ at the 3–5 leaf stage can beused and may be followed with a pre- or post-harvest application,depending on the type of wheat to be tested. The test parameters can beoptimized for each crop in order to find the particular plant comprisingthe constructs of the present invention that confers the desiredcommercially effective glyphosate tolerance level.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES Example 1

The plasmid constructs used are either pUC cloning constructs or doubleborder plant transformation constructs containing an E. coli origin ofreplication such as ori322, a broad host range origin of replicationsuch as oriV or oriRi, and a coding region for a selectable marker suchas Spc/Str that encodes for Tn7 aminoglycoside adenyltransferase (aadA)confers resistance to spectinomycin or streptomycin, or a gentamicin(Gm, Gent) selectable marker. For plant transformation, the hostbacterial strain was Agrobacterium tumefaciens ABI or LBA4404.

The genetic elements are described as follows: P-e35S is the 355 RNAfrom CAMV containing a duplication of the −90–300 region as described inU.S. Pat. No. 5,424,200 herein incorporated by reference in itsentirety; P-FMV is the 34S promoter from Figwort Mosaic Virus asdescribed in U.S. Pat. No. 5,378,619 herein incorporated by reference inits entirety; P-eFMV is a derivative of the FMV promoter containing aduplicated FMV promoter; CTP2 is the transit peptide region ofArabidopsis EPSP synthase as described in U.S. Pat. No. 5,633,435;aroA:CP4syn (aroA:CP4) is the coding region for CP4 EPSP (syntheticsequence) as described in U.S. Pat. No. 5,633,435 or further modifiedfor expression in plants based on codon usage of particular plantspecies; E9 3′ is the 3′ end of an isolate of the pea RbcS gene thatfunctions as a polyadenylation signal; nos is the 3′ end of the nopalinesynthase gene that functions as a polyadenylation signal; Hsp70 is thenon-translated leader sequence from Petunia hybrida as described in U.S.Pat. No. 5,362,865 herein incorporated by reference in its entirety; GUSis the beta-glucuronidase coding sequence from E. coli (Jefferson, R. A.Proc. Natl. Acad Sci. U.S.A., 83: 8447–8451, 1987); the right border(RB) and left borders (LB) are from the Ti plasmid of Agrobacteriumtumefaciens octopine and nopaline strains. The P-AtAct2 is the promoterfrom the Arabidopsis thaliana actin 2 gene; AtAct2i is the intron in the5′ untranslated region (UTR) of the Arabidopsis thaliana actin 2 gene;P-AtAct8 is the promoter from the Arabidopsis thaliana actin 8 gene;AtAct2i is the intron in the 5′ UTR of the Arabidopsis thaliana actin 8gene; P-AtAct11 is the promoter from the Arabidopsis thaliana actin 11gene; AtAct11 is the intron in the 5′ UTR of the Arabidopsis thalianaactin 11 gene; P-AtAct1a is the promoter from the Arabidopsis thalianaactin 1a gene, L-AtAct1a is the untranslated leader and I-AtAct1a is theintron from the genomic DNA of the actin 1a gene; P-AtAct1b is thepromoter from the Arabidopsis thaliana actin 1b gene, L-AtAct1b is theuntranslated leader and I-AtAct1b is the intron from the genomic DNA ofthe actin 1b gene; P-AtAct3 is the promoter from the Arabidopsisthaliana actin 3 gene, L-AtAct3 is the untranslated leader and I-AtAct3is the intron from the genomic DNA of the actin 3 gene; P-AtAct7 is thepromoter from the Arabidopsis thaliana actin 7 gene, L-AtAct7 is theuntranslated leader and I-AtAct7 is the intron from the genomic DNA ofthe actin 7 gene; P-AtAct12 is the promoter from the Arabidopsisthaliana actin 12 gene, L-AtAct12 is the untranslated leader andI-AtAct12 is the intron from the genomic DNA of the actin 12 gene;P-AtEF1α (P-AtEF1 or EF1α) is the promoter from the Arabidopsis thalianaelongation factor gene 1α, AtEF1α-i (AtEF1-i) is the intron in the 5′UTR of the Arabidopsis thaliana elongation factor gene 1α.

FIGS. 1–18 provide examples of plant transformation constructs thatcontain one to three plant expression cassettes. Multiple combinationsof plant expression cassettes comprising the promoter and geneticelements of the present invention can be constructed and tested in cropsplants by those skilled in the art of plant molecular biology withoutundue experimentation. The constructs illustrated in the Figures are notto be construed as the only constructs that can be assembled, but serveonly as examples to those skilled in the art. FIG. 1 (pCGN8086) providesan example of a plant transformation construct containing one expressioncassette comprising one promoter of the present invention (P-AtAct8)operably linked to a gene of interest (CTP2-aroA:CP4syn). FIG. 2(pMON45325) provides an example of a plant transformation constructcontaining two expression cassettes comprising at least one promoter ofthe present invention (P-AtAct11) operably linked to at least one geneof interest (CTP2-aroA:CP4syn). FIG. 3 (pMON45331) provides an exampleof a plant transformation construct containing one expression cassettecomprising one promoter of the present invention (P-AtEF1 plus intron)operably linked to at least one gene of interest (CTP2-aroA:CP4syn).FIG. 4 (pMON45332) provides an example of a plant transformationconstruct containing two expression cassettes comprising at least onepromoter of the present invention (P-AtEF1 plus intron) operably linkedto at least one gene of interest (CTP2-aroA:CP4syn). FIG. 5 (pMON9190)provides an example of a plant transformation construct containing threeexpression cassettes wherein at least two promoters of the presentinvention (P-AtEF1 plus intron, AtEF1a-i; P-AtAct2 plus intron, AtAct2i)are operably linked to at least one gene of interest (CTP2-aroA:CP4syn)and the P-EFMV promoter operably lined to CTP2-aroA:CP4syn. FIG. 6(pMON9153) plant expression cassettes are identical to those illustratedin FIG. 4 (pMON45332), this plasmid map is illustrated for the purposeof identification of the expression cassettes for data shown on plantphenotype in the data tables shown in the specification. FIG. 7(pCGN8099) provides an example of a plant transformation constructcontaining two expression cassettes comprising hybrid promoters of thepresent invention, P-FMV-AtEF1α and P-e35S-AtAct8, driving transcriptionof the gene of interest (aroA:CP4syn). FIG. 8 (pCGN8088) provides anexample of a plant transformation construct containing two expressioncassettes comprising one promoter of the present invention, P-AtAct8plus intron, AtAct8i, and the P-eFMV promoter driving expression of agene of interest (aroA:CP4syn). FIG. 9 (pCGN8068) provides an example ofa plant transformation construct containing two expression cassettescomprising one promoter of the present invention, P-AtAct2 plus intron,AtAct2i, and the P-eFMV promoter driving expression of a gene ofinterest (aroA:CP4syn). FIG. 10 (pCGN8096) provides an example of aplant transformation construct containing two expression cassettescomprising hybrid promoters of the present invention, P-FMV/AtAct11 andP-e35S-AtAct2, driving transcription of the gene of interest(aroA:CP4syn). FIG. 11 (pCGN9151) provides an example of a planttransformation construct containing two expression cassettes comprisinghybrid promoters of the present invention, P-FMV-AtEF1α andP-e35S-AtAct2, driving transcription of the gene of interest(aroA:CP4syn). FIG. 12 (pMON10156) provides an example of a planttransformation construct containing one expression cassette comprisingthe P-eFMV promoter driving expression of the aroA:CP4syn gene ofinterest, this vector is used for comparative purposes with the promotersequences of the present invention. FIG. 13 (pMON52059) provides anexample of a plant transformation construct containing one expressioncassette comprising a hybrid promoter (P-FMV-AtEF1α) driving theexpression of the gene of interest (aroA:CP4syn). FIG. 14 (pMON54952)provides an example of a plant transformation construct containing oneexpression cassette comprising one promoter of the present invention(P-AtAct1a plus AtAct1a intron) operably linked to at least one gene ofinterest (CTP2-aroA:CP4syn). FIG. 15 (pMON54953) provides an example ofa plant transformation construct containing one expression cassettecomprising one promoter of the present invention (P-AtAct1b plus AtAct1bintron) operably linked to at least one gene of interest(CTP2-aroA:CP4syn). FIG. 16 (pMON54954) provides an example of a planttransformation construct containing one expression cassette comprisingone promoter of the present invention (P-AtAct3 plus AtAct3 intron)operably linked to at least one gene of interest (CTP2-aroA:CP4syn).FIG. 17 (pMON54955) provides an example of a plant transformationconstruct containing one expression cassette comprising one promoter ofthe present invention (P-AtAct7 plus AtAct7 intron) operably linked toat least one gene of interest (CTP2-aroA:CP4syn). FIG. 18 (pMON54956)provides an example of a plant transformation construct containing oneexpression cassette comprising one promoter of the present invention(P-AtAct12 plus AtAct12 intron) operably linked to at least one gene ofinterest (CTP2-aroA:CP4syn).

Example 2

The cloning constructs and GUS constructs are listed in Table 1. TheArabidopsis actin 2 promoter and intron (Genbank accession number U41998as described in An et al., Plant J. 10: 107–121, 1996) was isolatedusing Arabidopsis thaliana Landsberg erecta DNA as a template (Rogersand Bendich, Plant Mol. Biol. 5:69, 1998) using SEQ ID NO:1 (forwardprimer) and SEQ ID NO:2 (reverse primer) in a reaction as follows: 0.5μg template DNA, 25 pmole of each primer, taq polymerase (BMB,Indianapolis, Ind.) using wax beads for “hot start” PCR. The PCRthermocycler conditions were as follows: 94° C. for one minute; 30cycles of: 92° C. for 40 seconds, 55° C. for one minute, 72° C. for oneminute and 30 seconds; and a five minute 72° C. extension. The PCRreaction was purified using GeneClean II (Bio101 Inc., Vista, Calif.),digested with HindIII and NcoI, and ligated into construct pMON26149(Table 1) digested with HindIII and NcoI. The promoter clone wassequence verified and the resulting construct was designated pMON26170(Table 1).

TABLE 1 Cloning Constructs and GUS Constructs containing ArabidopsisActin and EF1 promoter sequences Construct Description Promoter*/Gene/3′pMON26149 cloning construct pMON26170 plant expression constructAct2/GUS/nos pMON26171 plant expression construct Act8/GUS/nos pMON8677cloning construct pMON48407 plant expression construct Act11/GUS/nospMON26152 cloning construct pMON26177 plant expression constructEF1/GUS/nos pMON11750 plant expression construct e35S/GUS/nos pMON15737plant expression construct FMV/GUS/nos *the actin and elongation factorpromoter sequences also contain the intron sequence from the 5′ UTR ofthe corresponding gene.

Example 3

The Arabidopsis actin 8 promoter and intron (Genbank accession numberU42007 as described in An et al., Plant J. 10:107–121, 1996) wasisolated using Arabidopsis thaliana Landsberg erecta DNA as a templatePCR conditions and purification methods' described in Example 2 usingprimers SEQ ID NO:3 (forward primer) and SEQ ID NO:4 (reverse primer).The promoter was cloned using restriction enzymes as described inExample 2, sequence verified, and the resulting construct was designatedpMON26171 (Table 1).

Example 4

The Arabidopsis actin 11 promoter and intron (Genbank accession numberU27981 as described in Huang et al., Plant Mol. Biol., 33:125–139, 1997)was isolated using Arabidopsis thaliana Landsberg erecta DNA as atemplate PCR conditions and purification methods described in Example 2using primers SEQ ID NO:5 (forward primer) and SEQ ID NO:6 (reverseprimer). The promoter was cloned using restriction enzymes EcORV andNcoI and ligated into pMON8677 (Table 1), sequence verified, and theresulting construct was designated pMON48407 (Table 1).

Example 5

The Arabidopsis elongation factor 1α (AtEF1α) promoter and intron(Genbank accession number X 16430 as described in Axelos et al., Mol.Gen. Genet. 219:106–112, 1989; Curie et al., NAR 19:1305–1310; Curie etal., Plant Mol. Biol. 18:1083–1089, 1992; Curie et al., Mol. Gen. Genet.238:428–436, 1993) was isolated using Arabidopsis thaliana Landsbergerecta DNA as a template PCR conditions and purification methodsdescribed in Example 2 using primers SEQ ID NO:7 (forward primer) andSEQ ID NO:8 (reverse primer). The promoter was cloned using restrictionenzymes HindIII and NcoI and ligated into pMON26152 (Table 1) asdescribed in Example 2, sequence verified, and the resulting constructwas designated pMON26177 (Table 1).

Example 6

The plant transformation constructs described were mated intoAgrobacterium. Cotton transformation was performed essentially asdescribed in WO/0036911, herein incorporated by reference in itsentirety. The Arabidopsis transformation was performed as described inYe et al., Plant Journal 19:249–257, 1999. The tomato transformation wasperformed as described in U.S. Pat. No. 5,565,347 herein incorporated byreference in its entirety.

Example 7

A DNA construct is transformed into a target crop of interest via anappropriate delivery system such as an Agrobacterium-mediatedtransformation method (see for example U.S. Pat. No. 5,569,834 hereinincorporated by reference in its entirety, U.S. Pat. No. 5,416,011herein incorporated by reference in its entirety, U.S. Pat. No.5,631,152 herein incorporated by reference in its entirety, U.S. Pat.No. 5,159,135 herein incorporated by reference in its entirety, U.S.Pat. No. 5,004,863 herein incorporated by reference in its entirety, andU.S. Provisional Appln. No. 60/111,795 herein incorporated by referencein its entirety. Alternatively, a particle bombardment method may beused (see for example Patent Applns. WO 92/15675. WO 97/48814 andEuropean Patent Appln. 586,355, and U.S. Pat. Nos. 5,120,657, 5,503,998,5,830,728 and 5,015,580, all of which are herein incorporated byreference in their entirety).

A large number of transformation and regeneration systems and methodsare available and well-known to those of skill in the art. The stablytransformed plants and progeny are subsequently analyzed for expressionof the gene in tissues of interest by any number of molecular,immunodiagnostic, biochemical, and/or field evaluation methods known tothose of skill in the art, including, but not limited to a spray testwith a glyphosate formulation at commercially effective concentrationsperformed in a growth chamber or field environment.

Example 8

The GUS assays are performed by routine methods known to those of skillin the art (see for example, Jefferson et al., EMBO J. 6:3901, 1987).For cotton, R0 plants were tested. The tissue was size selected atvarious stages in development, samples and pooled for analysis. Thecotton floral bud was harvested and the male reproductive tissue samples(anthers and filaments), female reproductive tissue samples (entirestigma, style, and ovary), and corolla (sepals and petals) were taken.For the size selection, three floral buds from each stage were selectedthat included several sizes including small (less than 0.5 cm), medium(from 0.5–0.7 cm), and large (candle stage or open flower). Leaf sampleswere collected about 1–2 weeks after the cotton plants were placed inthe greenhouse, and the other samples were collected approximately 1–2months later. The first flowers were not collected (the first fivefruiting positions were left intact).

For Arabidopsis, V1 plants were analyzed and only homozygous andheterozygous segregants were tested Eight to ten events per constructwere analyzed (five plants per event). The GUS results for Arabidopsisrepresent pooled samples of 8–10 events. The values in the disclosedtables (Table 2 and Table 3) represent the average GUS expression forthe designated tissue (pmol/MU/min/mg).

Example 9

Plants were analyzed for GUS expression in leaf tissue and reproductivetissues including immature floral buds and flowers. The results areshown in Table 2. Constructs tested included pMON48407(P-AtAct11+intron/GUS/nos), pMON26170 (P-AtAct2+intron/GUS/nos),pMON26171 (P-AtAct8+intron/GUS/nos), pMON11750 (e35S/GUS/nos), pMON26177(P-EF1a+intron/GUS/nos), and pMON15737 (P-FMV/GUS/nos). The actin andelongation factor promoters conferred high levels of GUS expression inmultiple tissues including reproductive tissues.

TABLE 2 Average Arabidopsis V1 GUS Expression Immature Construct LeafFloral Bud Flower Gynoecium Andorecium pMON48407 6944 7394 8359 ND NDpMON26170 45238 74099 54502 73623 217292 pMON26171 29343 35884 3712576311 207100 pMON11750 60844 14032 16263 35882 115049 pMON26177 4759872871 96420 191066 507370 pMON15737 28314 57903 84457 44696 87876

Example 10

The R0 cotton plants were tested for expression of the GUS reporter genein selected tissues of various stages of development. The floral budswere staged by size (small, medium, and large; large=candle and openflower). The androecium represented the male reproductive tissuesincluding the entire receptacle (stigma, style, and ovaries). Thecorolla sample was composed of sepals and petals. The tissue wasprepared and GUS assays performed as described in EXAMPLE 8. The resultsare summarized in Table 3. The constructs tested included pMON48407(P-EF1α+intron/gus/nos), pMON26170 (P-AtAct2+intron/gus/nos), andpMON48407 (P-AtAct11+intron/gus/nos).

Six plants were tested and average GUS values obtained for pMON26177.Twenty plants were tested and average GUS values obtained for pMON26170.Eight plants were tested and average GUS values obtained for pMON48407.The results demonstrate that the actin and elongation factor promoterscan be used for effective expression of operably linked genes,particularly in reproductive tissues

TABLE 3 GUS Assay Results for Cotton Plants Construct Promoter/intronTissue Tested GUS Results pMON26177 EF1α Leaf 11600 pMON26177 EF1α SmallCorolla 396 pMON26177 EF1α Small Gynoecium 8670 pMON26177 EF1α SmallAndroecium 13771 pMON26177 EF1α Medium Corolla 362 pMON26177 EF1α MediumGynoecium 3318 pMON26177 EF1α Medium Androecium 8006 pMON26177 EF1αLarge Corolla 351 pMON26177 EF1α Large Gynoecium 500 pMON26177 EF1αLarge Androecium 15512 pMON26170 Act2 Leaf 12718 pMON26170 Act2 SmallCorolla 1296 pMON26170 Act2 Small Gynoecium 16684 pMON26170 Act2 SmallAndroecium 7570 pMON26170 Act2 Medium Corolla 742 pMON26170 Act2 MediumGynoecium 10041 pMON26170 Act2 Medium Androecium 7893 pMON26170 Act2Large Corolla 289 pMON26170 Act2 Large Gynoecium 3218 pMON26170 Act2Large Androecium 42737 pMON48407 Act11 Leaf 28289 pMON48407 Act11 SmallCorolla 10 pMON48407 Act11 Small Gynoecium 40755 pMON48407 Act11 SmallAndroecium 47834 pMON48407 Act11 Medium Corolla 742 pMON48407 Act11Medium Gynoecium 52495 pMON48407 Act11 Medium Androecium 35573 pMON48407Act11 Large Corolla 1072 pMON48407 Act11 Large Gynoecium 4869 pMON48407Act11 Large Androecium 42737

Example 11

Transformed plants were also tested in a greenhouse spray test usingRoundup Ultra™ a glyphosate formulation with a Track Sprayer device(Roundup Ultra is a registered trademark of Monsanto Company). Plantswere at the “two” true leaf or greater stage of growth and the leaveswere dry before applying the Roundup® spray. The formulation used wasRoundup Ultra™ as a 3 lb/gallon a.e. (acid equivalent) formulation. Thecalibration used was as follows:

For a 20 gallons/Acre spray volume:

Nozzle speed: 9501 evenflow Spray pressure:  40 psi Spray height  18inches between top of canopy and nozzle tip Track Speed 1.1 ft/sec.,corresponding to a reading of 1950 - 1.0 volts. Formulation: RoundupUltra ™ (3 lbs. A.e./gallon)

The spray concentrations will vary, depending on the desired testingranges. For example, for a desired rate of 8 oz/acre a working solutionof 3.1 ml/L is used, and for a desired rate of 64 oz/A a working rangeof 24.8 ml/L is used.

The evaluation period will vary, depending on the crop, stage of plantdevelopment, and tolerance level desired.

Example 12

The plant expression constructs used for tomato transformation arelisted in Table 4. Tomato plants (T0) containing constructs comprisingat least one actin or elongation factor promoter (with intron) operablylinked to an aroA:CP4 glyphosate tolerance gene are screened in agreenhouse glyphosate spray test with glyphosate (Roundup Ultra™)formulation for the efficiency of conferring glyphosate tolerance totransgenic tomato plants. Optionally, at least one actin or elongationfactor promoter sequence operably linked to an aroA:CP4 gene and an eFMVcaulimovirus promoter operably linked to an aroA:CP4 transformed intotomato plants are screened by spray application with glyphosate (RoundupUltra™). Tomato plants are sprayed with 48 oz./acre then evaluated attwo weeks post application for analysis of vegetative tolerance and upto 60 days post-application for analysis of reproductive tolerance. Theresults are shown in Table 4 and ranked according to efficiency ofselecting reproductive tolerant lines. The percent vegetative toleranceis the percentage of the lines screened that demonstrated sufficientvegetative tolerance to glyphosate damage to be considered for furtherstudies of agronomic traits in preparation for commercially candidacy.The percent reproductive tolerance is the percentage of the vegetativetolerant lines that also demonstrated sufficient reproductive toleranceto be considered for further agronomic evaluation. All of the constructsproved functional for providing vegetative tolerance and reproductivetolerance to the transgenic tomato plants. Various combinations ofpromoters are able to increase the efficiency at which vegetative andreproductive tolerant lines could be selected by screening in thisexperiment. Constructs containing the Arabidopsis EF1α promoter are morespecifically associated with a high percentage of vegetatively tolerantlines. P-Act2 promoter in combination with P-eFMV and P-AtEF1α(pCGN9190) provided an increase in the percentage of reproductivelytolerant lines that are screened by this method.

TABLE 4 Greenhouse Track Spray Trials with Application Rate of 48oz./Acre* # Lines % Vegetative % Reprod. Construct Description TestedTolerance¹ Toler.² pCGN9190 eFMV/CP4 + EF1α/CP4 + Act2/CP4 930 83.2 52.4pCGN9153 EF1α/CP4 + eFMV/CP4 391 73.9 38.9 pCGN8086 Act8/CP4 21 47.638.1 pCGN8099 FMV-EF1α/CP4 + Act8/CP4 71 84.5 36.6 pCGN8088 eFMV/CP4 +Act8/CP4 144 79.9 34.7 pMON45325 eFMV/CP4 + Act11/CP4 90 70.0 34.4pCGN8096 FMV-Act11/CP4 + Act2/CP4 201 62.7 10.4 pCGN8067 Act2/CP4 20567.3 8.8 *one application ¹Pooled Results from 25 screens. Scored 14days post-application ²Pooled Results from 25 screens. Scored up to 60days post-application

Tomato seed yield is used as a measure of the efficacy of the variouspromoter sequences and combination of expression cassettes used in thepresent invention for conferring glyphosate tolerance to transgenictomato plants. In Table 5, the results of three field experiments areshown on transgenic tomato plants containing constructs with thepromoters of the present invention driving expression of the aroA:CP4coding sequence for glyphosate tolerance. Experiment 1 is a test of theplants produced from the constructs that contain the Figwort mosaicvirus promoter (P-FMV) in the native and the duplicated version (P-eFMV)and additional genetic elements in the constructs that are also found inthe constructs used to test the promoter sequences of the presentinvention. Additional genetic elements such as the source of the 5′untranslated sequence and the chloroplast transit peptide are alsotested. The construct pMON20998 comprises the P-eFMV, linked to thepetunia Hsp70 5′ UTR, leader linked to the Arabidopsis EPSPS chloroplasttransit peptide (CTP2), linked to the E9 3′ termination region. Theconstruct pMON20999 differs from pMON20998 only in that the promoter isP-FMV. The construct pMON10156 differs from pMON20998 only in that theCTP is from the Petunia EPSPS chloroplast transit peptide (CTP4). Theconstruct pMON45312 differs from pMON20998 only in that the leadersequence is the native FMV leader sequence.

Tomato plants are transplanted into the field in rows. The plants arespray treated in the field at a rate of 48 oz./Acre with Roundupherbicide. The tomato seed is collected from the fruit and weighted. Anunsprayed tomato line serves as the control for comparison purposes andthe efficacy of each construct is expressed as a percentage of thecontrol. The result of Experiment 1 (column 1 of Table 5) is that theFMV promoter and P-eFMV only provide 5–11% of the seed production of anunsprayed check. Experiment 2, and 3 tests the constructs of the presentinvention at different locations (columns 2 and 3 of Table 5).Experiment 2 is conducted at the same location as Experiment 1, theconstructs pCGN8099 (FIG. 7), pCGN9151 (FIG. 11) and pCGN9190 (FIG. 5)performed well by providing 25–46% of the seed relative to an unsprayedcheck. At a different location that has a cooler growing season,Experiment 3 demonstrated that pCGN8068 (FIG. 9), pCGN8088 (FIG. 8),pCGN8099, pCGN9151, pCGN9153 (FIG. 6), and pMON45325 (FIG. 2) are ableto confer sufficient glyphosate tolerance for the tomatoes to set 34–77%of normal seed set relative to an unsprayed check.

TABLE 5 Tomato seed yield experiments Exp. 2 Exp. 1 Seed Exp. 3 Seed wt% of wt % of Seed wt % of grams Control grams Control grams ControlpMON20998 0.52 5.3 pMON20999 0.84 8.6 pMON10156 0.50 5.1 pMON45312 1.0711.0 pCGN8068 0.48 8.4 7.06 77.8 pCGN8088 0.43 7.6 3.09 34.1 pCGN80960.40 7.0 pCGN8099 1.85 32.5 6.93 76.4 pCGN9151 1.46 25.7 6.11 67.4pCGN9153 0.68 12.0 4.03 44.4 pCGN9190 2.64 46.4 pMON45325 0.31 5.4 3.3737.2 pCGN8067 Control 9.73 100.0 5.69 100.0 9.07 100.0

Example 13

SEQ ID NOS: 1–8, and SEQ ID NOS:13–21 are PCR primers designed frompublicly available sequence information for Arabidopsis thaliana Act1,Act2 (Genbank #U41998), Act3, Act7, Act8 (Genbank #ATU42007), Act11(Genbank #ATU27981), Act12 and Elf1α (Genbank #X16430) genes. Thesesequences are used to extend the nucleic acid sequence using polymerasechain reaction (PCR) amplification techniques (see for example, Mulliset al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1986; Erlich, etal., European Patent Appln. 50,424; European Patent Appln. 84,796,European Patent Appln. 258,017, European Patent Appln. 237,362; Mullis,European Patent Appln. 201,184; Mullis, et al., U.S. Pat. No. 4,683,202;Erlich, U.S. Pat. No. 4,582,788; and Saiki, et al., U.S. Pat. No.4,683,194). A number of PCR amplification methods are known to those ofskill in the art and are used to identify nucleic acid sequencesadjacent to a known sequence. For example, inverse PCR (IPCR) methods,which are used to amplify unknown DNA sequences adjacent to a coreregion of known sequence have been described. Other methods are alsoavailable such as capture PCR (Lagerstrom M., et al., PCR MethodsApplic. 1: 111, 1991), and walking PCR (Parker, et al., Nucleic AcidsRes 19:3055, 1991). A number of manufacturers have also developed kitsbased on modifications of these methods for the purposes of identifyingsequences of interest. Technical advances including improvements inprimer and adaptor design, improvements in the polymerase enzyme, andthermocycler capabilies have facilitated quicker, efficient methods forisolating sequences of interest.

Table 5a. Primer Sequences for Isolation of Arabidopsis Actin and EF1αPromoter Sequences

TABLE 5a Primer sequences for isolation of Arabidopsis Actin and EF1αpromoter sequences SEQ Name ID NO: Sequence At. Actin 2 forward 1TTTTTTTTGA TATCAAGCTT CAACTATTTT TATGTATGC At. Actin 2 reverse 2GCCTCAGCCA TGGTGAGTCT GCTGCAAACA CACAAAAAGA GTTCAAT At. Actin 8 forward3 TTTTTTTTGA TATCAAGCTT CCATTTTTCT TTTGCATAAT TC At. Actin 8 reverse 4GCATCGGCCA TGGTGAGTCT TCTGCAATCA AAAACATAAA GATCTGA At. Actin 11 forward5 TTTTTTTTTA AGCTTGATAT CACAACCAAA TGTCAAATGG At. Actin 11 reverse 6CCATCTGCCA TGGTCTATAT CCTGTC At. EF1 α forward 7 TTTTTTTTTA AGCTTGATATCGGAAGTTTC TCTCTTG At. EF1 α reverse 8 CTTTTCCCAT GGTAGATCTC TGGTCAACAAATC At. Actin 1a forward 13 CCCAAGCTTA AATGACATCA GATACACGC At. Actin 1bforward 14 CATAAGCTTA GAGGTCCAAA TTCA At. Actin 1 reverse 15 CCATCAGCCATGGTCTTCTA CCTTTATGCA AA At. Actin 3 forward 16 CCAAGCTTAC CACACTCAGATGCATAAACA AACACA At. Actin 3 reverse 17 CATCAGCCAT GGTCTACTCTCTGCAAAAAC A At. Actin 7 forward 18 GCAAAGCTTA CTAGTCAACA ATTGGCC At.Actin 7 reverse 19 GATCGGCCAT GGTTCACTAA AAAAAAAG At. Actin 12 forward20 GGAAGCTTGC GGCCGCTTTC TACTCTACAT GTTTCT At. Actin 12 reverse 21GACTAGCCGC CATGGTTCAA TCTCTAGCTG A

The leaves of young plants of Arabidopsis thaliana (1 g) werehomogenized in 9 ml of CTAB buffer (Saghai-Maroof et al. 1984, PNAS81:8014–8018). The CTAB buffer contained 100 mM Tris HCl, pH 7.8, 700 mMNaCl, 50 mM EDTA, 1% CTAB (alkytrimethyhyl-ammoniumbromide) and 140 mM2-mercaptoethanol. After 90 minutes incubation in 65 C, 4.5 ml ofchloroform:isoamyl alcohol (24:1) was added and samples were mixed for10 minutes. Aqueous layer was separated by centrifugation for 10 minutesat 1500 g and was re-extracted with chloroform:isoamyl alcohol. Aftersecond centrifugation, aqueous layer was transferred to a tubecontaining 50 μl 10 mg/ml RNase A (DNase free) and incubated in roomtemperature for 30 minutes to remove RNA. DNA was precipitated with 6 mlof isopropanol and re-suspended in 1 ml of 10 mM Tris HCl buffer pH 8.5.DNA solution was extracted once with equal volume of phenol and oncewith an equal volume of chloroform: isoamylalcohol. Aftercentrifugation, 1/10 volume of sodium acetate (3M, pH 5.2) was added toaqueous layer, followed by 2.5 volume of ethanol. The DNA was hooked,washed in 70% ethanol, then air dried and re-suspended in 0.2 ml of 10mM Tris HCl buffer.

Arabidopsis genomic DNA (100 ng) was used in 50 ml PCR reactions.Reactions containing the primers shown in Table 5a. contained 0.2 mMreverse and forward primer solutions, 200 nM dNTPs and PCR buffer withmagnesium and DNA polymerase mix from Expand™ High Fidelity PCR System(Roche Molecular Biochemicals). After initial 2 minute denaturation at94° C. reactions were cycled 0.5 mm at 94° C., 0.5 mm at 55° C. and 1.5minute at 72° C. for 35 times. PCR products were analyzed byelectrophoresis on 1% agarose gel. Gel isolated DNA fragmentsrepresenting Actin 1a, Actin 1b, Actin 7, and Actin 12 sequences werephosphorylated with T4 DNA kinase and ligated to dephosphorylated andSma I cut pUC19 cloning construct. White colonies were screened for thepresence of appropriate inserts and sequenced with M13 to confirm thepresence of actin promoters. Selected clones were designated aspMON54941 (P-AtAct1a), pMON54942(P-AtAct1b), pMON54943 (P-AtAct7) andpMON54944 (P-AtAct12). Subsequently, the Actin promoters DNA fragmentswere released by Hind III and NeoI digest of the pUC19 constructscontaining the insert sequences, the DNA fragments were gel isolated andligated to pMON26165 that had been digested with the same restrictionenzymes. A PCR product for the Actin 3 promoter (P-AtAct3) was digestedwith Hind III and Nco I and cloned directly into pMON26165 to formpMON54951. pMON26165 contains the GUS/nos terminator gene segment.Ligation with the promoter segments allows for assay of each promoterfor functional activity by expression of the β-glucuronidase enzyme inplant cells. The plant cells can be isolated, for example, tobacco leafprotoplasts, or the plant cells may be contained in a plant tissue ororgan, such as, leaf, root, cotyledon, hypocotyl, embryo, flower, orstorage organ.

The constructs containing aroA:CP4 EPSPS driven by the Arabidopsis Actin1a, (pMON54952), Actin 1b (pMON54953), Actin 3 (pMON54954), Actin 7(pMON54955) and Actin 12 (pMON54956) promoters of the present inventionwere prepared in Agrobacterium binary plant transformation constructsfor stable expression of the glyphosate resistant EPSPS in crop plants.These constructs are transformed into soybean and cotton cells, thecells are selected and regenerated into plants on glyphosate containingtissue culture media and then assayed for expression of the aroA:CP4protein and for tolerance to glyphosate application. Plantsdemonstrating commercially acceptable glyphosate tolerance are furtherdeveloped by conventional breeding methods to transfer the glyphosatetolerance trait into germplasm adapted for cultivation.

TABLE 6 Activity of different Arabidopsis actin promoters in transientassay as compare to P-e35S. Construct GUS Activity Pe35S/GUS +++P-AtAct1a/GUS ++ P-AtAct1b/GUS ++ P-AtAct3/GUS ++ P-AtAct7/GUS ++P-AtAct12/GUS +

Example 14

Cotton yield is correlated with the number of squares set during thefirst four to five weeks of squaring. The retention of these squares tomature bolls and their contribution to the harvest of the cotton lint isa key component of yield. When determining the efficacy of transgeneconstructs for conferring herbicide tolerance in cotton, the amount ofboll retention is a measure of efficacy and is a desirable trait.Transgenic cotton plants containing promoters of the present invention(Table 7) were assayed in greenhouse conditions for boll retention. Thepromoters directed expression of the aroA:CP4 coding sequence forglyphosate tolerant phenotype. The plants were transformed by anAgrobacterium-mediated method or by a particle gun method. The particlegun constructs contained an additional GUS containing expressioncassette useful for histochemical localization of β-glucuronidaseactivity from the promoters of the present invention. Transgenic plantswere regenerated on glyphosate containing media and plants rooted on arooting media. The rooted plantlets were potted in soil and transferredto a growth chamber for a hardening off period. The seed from theseplant lines were collected and planted. Fifteen plants from each linewere sprayed with glyphosate at 48 ounces/acre at the 4 leaf stage. Atleast 8 surviving plants from each line were sprayed again at the 8 leafstage with glyphosate at 48 ounces/acre. At maturity, the number offirst position bolls for the first five bolls was counted. Those linesthat had 3 or more of the first position bolls retained after theglyphosate spray (plant map≧3) were advanced for further study. Table 7illustrates the data produced from this study. The number of linesmapped indicates the number of lines surviving the first glyphosatespray application. The commercial standard is Line 1445 (pMON 17136)that contains the P-FMV promoter driving expression of the CTP2-aroA:CP4gene/E9 3′, this line retains less than 1 of the 5 first bolls. Theconstructs, pCGN8099, pCGN9153, pCGN8088, pCGN8068 provided sufficientreproductive glyphosate tolerance in cotton such that 14–35% of thelines tested from these constructs were advanced for further agronomictrials.

TABLE 7 Greenhouse cotton boll retention study # lines Plant ConstructPromoters Mapped Map ≧ 3 % ≧ 3 pCGN8099 FMV:EF1α + e35S:Act8 104 3634.6% pCGN9153 EF1α + FMV 36 12 33.3% pCGN9165 EF1α + 35S/GUS 3 1 33.3%pCGN9152 EF1a 7 0 0.0% pCGN8088 Act8 + FMV 43 6 14.0% pCGN8086 Act8 7 00.0% pCGN8068 Act2 + FMV 37 7 18.9% pCGN8067 Act2 37 0 0.0% pCGN8084Act2 + FMV + 35S/GUS 5 0 0.0% pCGN8085 Act2 + FMV/GUS 1 0 0.0% pCGN9164Act11 + 35S/GUS 21 1 4.8% pMON45325 Act11 + FMV 43 0 0.0% pCGN8096FMV:Act11 + e35S:Act2 14 0 0.0% pCGN9154 FMV:Act11 + e35S:Act2 16 1 6.3%Line 1445 FMV <1.0

Example 15

Cotton yield is correlated with the number of squares set during thefirst four to five weeks of squaring. The retention of these squares tomature bolls and their contribution to the harvest of the cotton lint isa key component of yield. When determining the efficacy of transgeneconstructs for conferring herbicide tolerance in cotton, the amount ofboll retention is a measure of efficacy and is a desirable trait.Transgenic cotton plants containing promoters of the present inventionwere assayed in field conditions at two locations for boll retention.The transgenic cotton lines 502–254–2 (pCGN8068), 701–178–2 (pCGN8068),53–2 (pCGN8088), 178–1 (pCGN9153), and 60–1 (pCGN9153) were compared to1445 (glyphosate tolerance line) and PM1218BR (Paymaster 1218 parent)that contain the construct pMON17136 (P-FMV/CTP2-aroA:CP4/E93′), a wildtype non-transgenic line, Coker 130 was included. The field design is arandomized complete block design consisting of 2 rows×20–30 feet×3replications.

Glyphosate is applied as Roundup Ultra™ formulation at rates of 1.12 lbai/A=48 oz product and 1.5 lb ai/A=64 oz product at the 8 leaf stage ofcotton plant development. All of the cotton plots are managedaggressively for weed and insect pest control, as well as otheragronomic inputs such as planting time, fertilization, irrigation, PGRusage and defoliation. The percent boll retention is determined bymapping the location of each of the retained bolls by random selectionof ten plants from the middle of the two center rows (five from eachrow) of each plot to map. The first mapping should be done 4 weeks afterfirst flower (mid-season map), a second mapping should be done atharvest. The data collected includes the number of first position bollson the bottom five flowering nodes that are counted as an indication ofthe reproductive tolerance of the transgenic cotton lines to glyphosate.Table 8 illustrates the advantage that promoters of the presentinvention have conferred to transgenic cotton plants for boll retention.This enhanced reproductive tolerance has resulted in increased lintyield (Table 9) and increased seed yield (Table 10) as well.

TABLE 8 Boll retention at mid-season plant map of bottom 5 firstposition bolls Location 1 Location 2 48 64 48 64 Untreated oz/A oz/AUntreated oz/A oz/A (17136) 1445 68 67 53 81 63 62 (8068) 502-254-2 8772 64 77 80 69 (8068) 701-178-2 85 77 60 84 86 76 (8088) 53-2 89 81 8079 76 73 (9153) 178-1 77 83 73 85 71 79 (9153) 60-1 80 89 81 77 82 87PM1218BR 92 56 63

TABLE 9 Lint Yield (lbs/Acre) and percent yield (Location 1) 64 oz/Cultivar Untreated 48 oz/A 48 oz/A % 64 oz/A A % 8068-502-254-2-4 1103960 87.0% 858 77.8% 8068-701-178-2-2 1326 1219 91.9% 1177 88.8%9153-60-1-1 1177 1206 102.5% 1171 99.5% 9153-178-1-1 1112 769 69.2% 75067.4% 8088-53-2-11 1283 1071 83.5% 1097 85.5% 1445 1018 563 55.3% 49048.1% C130 1200 0 0.0% 0 0.0% PM 1218 BR 1092 826 75.6% 713 65.3%

TABLE 10 Seed Cotton Yield (lbs/Acre) and percent yield (Location 1) 64oz/ Cultivar Untreated 48 oz/A 48 oz/A % 64 oz/A A % 8068-502-254-2-43357 2923 87.1% 2646 78.8% 8068-701-178-2-2 3720 3521 94.7% 3328 89.5%9153-60-1-1 3294 3413 103.6% 3316 100.7% 9153-178-1-1 3468 2355 67.9%2218 64.0% 8088-53-2-11 3404 2950 86.7% 2968 87.2% 1445 2835 1624 57.3%1372 48.4% C130 3272 0 0.0% 0 0.0% PM 1218 B/RR 3036 2192 72.2% 188562.1%

Example 16

The efficacy of the hybrid promoter P-FMV-AtEF1α driving expression ofthe CTP2-aroA:CP4 coding sequence (FIG. 13, pMON52059) andP-FMV/CTP2-aroA:CP4/E93′ (pMON15737) was compared in transgenicArabidopsis thaliana. The transgenic Arabidopsis thaliana plants wereproduced by the vacuum infiltration (Bechtold et al., C R Acad ParisLife Sci 316: 1194–1199) seeds were potted in soil in trays in a growthchamber adjusted for 24° C., 16 hour light (120 μE m⁻² s⁻¹) cycle topermit normal growth and development of the plants. The pMON52059 V1event glyphosate tolerant transgenic Arabidopsis plants were selected byspray application of glyphosate herbicide at a rate of 24 ounces/acre,the surviving plants were transplanted into individual pots. EightpMON52059 V1 plants and eight PMON 15737 homozygous plants were sprayeda second time corresponding to the observation of bolting, approximately16 days after the at a rate of 24 ounces/acre. The second spray willdetermine the efficacy of the two constructs for conferring reproductivetolerance. The plants were observed for vegetative effects of glyphosateapplication. All plants had complete vegetative tolerance and noabnormal flowers were observed. However, abortion of siliques occurredindicated that seed had not been set in the aborted siliques. The totalnumber of siliques produced by each plant and the siliques thatcontained seeds (fertile siliques) were counted and tabulated. Theresults are shown in Table 9 and indicate that the hybrid promoterconstruct pMON52059 demonstrated a greater than 10 fold improvement infertile siliques, 89% compared to pMON15737 at 8%. The number of fertilefruiting structures is related to the amount of seed that can beproduced, this is especially important in crops whose yield isassociated with seed numbers. Crops such as cotton, soybean, canola,wheat, and corn are crops where reproductive tolerance to glyphosate isessential for good yield.

Table 11. Comparison of the hybrid promoter P-FMV-EF1α (PMON52059) andP-FMV (PMON15737) in conferring reproductive tolerance to glyphosate inArabidopsis plants.

TABLE 11 Comparison of the hybrid promoter P-FMV-EF1α (pMON52059) andP-FMV (pMON15737) in conferring reproductive tolerance to glyphosate inArabidopsis plants. pMON52059 Plant pMON15737 Num- Fertile Total PercentPlant Fertile Total Percent ber Siliques Siliques Fertility NumberSiliques Siliques Fertility 8819 39 50 78.0% 1 74 540 13.7% 8820 626 69190.6% 2 23 600 3.8% 8821 507 561 90.4% 3 1 470 0.2% 8822 0 69 0.0% 4 20646 3.1% 8823 512 534 95.9% 5 43 717 6.0% 8827 326 354 92.1% 6 22 6513.4% 8833 432 461 93.7% 7 178 868 20.5% 8838 323 374 86.4% 8 40 520 7.7%Total 2765 3094 89.4% Total 401 5012 8.0%

Example 17

Sunflower (Helianthus annuus L.) is a crop of agronomic importance foroil and food. The constructs pMON45325 (FIG. 2), pMON45332 (FIG. 4), andpMON4533 1 (FIG. 3) of the present invention were transformed intosunflower. Agrobacterium-mediated transformation of sunflower has beenreported (Schrammeijer et al, Plant Cell Reports, 9: 55–60, 1990; EP 0486 234). Methods known by those skilled in the art of planttransformation with transgene expression constructs can includehypocotyls, apical meristems, protoplasm, and other sunflower tissues.Transgenic sunflower lines SFB250-27 contains pMON20999(P-FMV/CTP2-aroA:CP4/E93′) expression cassette; SFB288-01, SFB295-09contain pMON45325(P-FMV/CTP2-aroA:CP4/E93′::P-AtAct11+intron/CTP2-aroA:CP4/E93′);SFB289-01 contains pMON45332(P-AtEF1α+intron/CTP2-aroA:CP4/E93′::P-eFMV/CTP2-aroA:CP4/E93′);SFB303-08, SFB303-09, SFB303-11, and HA300B contain pMON45331(P-AtEF1α+intron/CTP2-aroA:CP4/E9). These lines are tested forglyphosate tolerance and are shown in Table 12.

The reproductive tolerance to glyphosate in sunflower can be measured asa function of the percent of normal heads, percent normal head size andthe pollen production. These plants are sprayed with Glyphosate at V-4and V-8 leaf stages at 0, 32 oz/acre or 64 ounces/acre rate. Thesunflower plants are assessed for vegetative tolerance to glyphosate.Vegetative tolerance is achieved at 32 and 64 oz/acre levels ofglyphosate spray at both V4 and V8 stages of plant development.

Vegetative glyphosate tolerant transgenic sunflower lines are scored fornumber of heads, percent normal heads, percent normal head size, andpercent normal pollen shed. These traits are scored in a field test atone location. The tabulation of the head scores and pollen production isshown in Table 12. Lines selected from the constructs of the presentinvention show greater percent of normal heads, generally greaterpercent normal head size and better pollen shed.

TABLE 12 Sunflower glyphosate resistance scores % normal % normal Line ## heads heads head size % pollen shed SFB250-27 28 29 75 36 SFB288-01 1136 73 73 SFB295-09 28 57 64 68 SFB289-01 13 38 92 38 SFB303-08 25 68 9264 SFB303-09 43 81 88 88 SFB305-11 45 71 84 100 HA300B 30 100 97 97non-trans segregant 0 0 0 0

Example 18

Cis acting regulatory elements necessary for proper promoter regulationcan be identified by a number of means. In one method, deletion analysisis carried out to remove regions of the promoter and the resultingpromoter fragments are assayed for promoter activity. DNA fragments areconsidered necessary for promoter regulation if the activity of thetruncated promoter is altered compared to the original promoterfragment. Through this deletion analysis, small regions of DNA can beidentified which are necessary for positive or negative regulation oftranscription. Promoter sequence motifs can also be identified and novelpromoters engineered to contain these cis elements for modulatingexpression of operably linked transcribable sequences. See for exampleU.S. Pat. No. 5,223,419, herein incorporated by reference in itsentirety, U.S. Pat. No. 4,990,607 herein incorporated by reference inits entirety, and U.S. Pat. No. 5,097,025 herein incorporated byreference in its entirety.

An alternative approach is to look for similar sequences betweenpromoters with similar expression profiles. Promoters with overlappingpatterns of activity can have common regulatory mechanisms. Severalcomputer programs can be used to identify conserved, sequence motifsbetween promoters, including but not limited to MEME, SIGNAL SCAN, orGENE SCAN. These motifs can represent binding sites for transcriptionsfactors which act to regulate the promoters. Once the sequence motifsare identified, their function can be assayed. For example, the motifsequences can be deleted from the promoter to determine if the motif isnecessary for proper promoter function. Alternatively, the motif can beadded to a minimal promoter to test whether it is sufficient to activatetranscription. Suspected negative regulatory elements can be tested forsufficiency by adding to an active promoter and testing for a reductionin promoter activity. Some cis acting regulatory elements may requireother elements to function. Therefore, multiple elements can be testedin various combinations by any number of methods known to those of skillin the art.

Once functional promoter elements have been identified, promoterelements can be modified at the nucleotide level to affect proteinbinding. The modifications can cause either higher or lower affinitybinding which would affect the level of transcription from thatpromoter.

Promoter elements can act additively or synergistically to affectpromoter activity. In this regard, promoter elements from different 5′regulatory regions can be placed in tandem to obtain a promoter with adifferent spectrum of activity or different expression profile.Accordingly, combinations of promoter elements from heterologous sourcesor duplication of similar elements or the same element can confer ahigher level of expression of operably linked transcribable sequences.For example, a promoter element can be multimerized to increase levelsof expression specifically in the pattern affected by that promoterelement.

The technical methods needed for constructing expression constructscontaining the novel engineered 5′ regulatory elements are known tothose of skill in the art. The engineered promoters are tested inexpression constructs and tested transiently by operably linking thenovel promoters to a suitable reporter gene such as GUS and testing in atransient plant assay. The novel promoters are operably linked to one ormore genes of interest and incorporated into a plant transformationconstruct along with one or more additional regulatory elements andtransformed into a target plant of interest by a suitable DNA deliverysystem. The stably transformed plants and subsequent progeny areevaluated by any number of molecular, immunodiagnostic, biochemical,phenotypic, or field methods suitable for assessing the desiredcharacteristic(s).

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. Transgenic cotton seed comprising one or more DNA expressionconstructs, said constructs comprising a hybrid promoter DNA sequencecomprising the 34S FMV promoter operably linked to an Arabidopsis EF1αpromoter; a structural DNA sequence encoding a glyphosate toleranceprotein; and a 3′ non-translated region that functions in plants tocause the addition of polyadenylated nucleotides to the 3′ end of theRNA sequence; wherein the structural DNA sequence is operably linked tothe hybrid promoter and the 3′ non-translated region.
 2. The transgeniccotton seed of claim 1, wherein the glyphosate tolerance protein is aglyphosate oxidoreductase (GOX) protein.
 3. The transgenic cotton seedof claim 1, wherein the glyphosate tolerance protein is an5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) protein.
 4. Thetransgenic cotton seed of claim 3, wherein the glyphosate toleranceprotein is aroA:CP4 EPSPS.
 5. The transgenic cotton seed of claim 1,wherein the DNA sequence encoding the glyphosate tolerance protein isfurther operably linked to a structural DNA sequence encoding achloroplast transit peptide.
 6. The transgenic cotton seed of claim 1,wherein the FMV promoter comprises a duplicated enhancer (eFMV).
 7. Thetransgenic cotton seed of claim 1, wherein the hybrid promoter is SEQ IDNo:
 28. 8. A regenerated transgenic cotton plant from the seed of anyone of claims 1–5, 6 and
 7. 9. The regenerated transgenic cotton plantof claim 8, further characterized as having a boll map value greaterthan or equal to 3 following exposure to glyphosate application afterthe 4 leaf stage.
 10. A transgenic cotton plant transformed with anucleic acid molecule comprising a structural DNA sequences encoding aglyphosate tolerance protein operably linked to a hybrid promotercomprising the 34S FMV promoter operably linked to an Arabidposis EF1apromoter which hybrid promoter drives sufficient expression of theprotein in the plant cells to provide vegetative reproductive glyphosatetolerance to the plant, said plant characterized as being capable ofhaving a boll map value greater than or equal to 3 following exposure toglyphosate after the 4 leaf stage.
 11. The transgenic cotton plant ofclaim 10, wherein the boll map value is determined by spraying thecotton plant after the 4 leaf stage with glyphosate at 48 ounces/acre,allowing the cotton plant to grow to maturity, and determining thenumber of first position bolls for the first five bolls to determine theboll map value.
 12. The transgenic cotton plant of claim 10, wherein theFMV promoter comprises a duplicated enhancer (eFMV).
 13. The transgeniccotton plant of claim 10, wherein the hybrid promoter is SEQ ID No: 28.14. The transgenic cotton plant of claim 10, wherein the glyphosatetolerance protein is a glyphosate oxidoreductase (GOX) protein.
 15. Thetransgenic cotton plant of claim 10, wherein the glyphosate toleranceprotein is an 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) protein.16. The transgenic cotton plant of claim 15, wherein the glyphosatetolerance protein is aroA:CP4 EPSPS.
 17. The transgenic cotton plant ofclaim 10, wherein the nucleic acid molecule further comprises astructural DNA sequence encoding a chloroplast transit peptide operablylinked to the DNA sequence encoding the glyphosate tolerance protein.